The biology of human longevity -  inflammation, nutrition, and aging in the evolution of lifespans
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Great comprehensive coverage of aging for healthcare providers, nutritionists, and experts in the medical fields.

Great comprehensive coverage of aging for healthcare providers, nutritionists, and experts in the medical fields.

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The biology of human longevity -  inflammation, nutrition, and aging in the evolution of lifespans The biology of human longevity - inflammation, nutrition, and aging in the evolution of lifespans Document Transcript

  • THE BIOLOGY OF HUMAN LONGEVITY
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  • THE BIOLOGY OF HUMAN LONGEVITY Inflammation, Nutrition, and Aging in the Evolution of Life Spans Caleb E. Finch Davis School of Gerontology and USC College University of Southern California AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier
  • Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WCIX 8RR, UK This book is printed on acid-free paper. Copyright © 2007, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: permissions@elsevier.com. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Support & Contact” then “Copyright and Permission” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data Finch, Caleb Ellicott. The biology of human longevity : inflammation, nutrition, and aging in the evolution of lifespans / Caleb E. Finch. p. cm. Includes bibliographical references and index. ISBN-13: 978-0-12-373657-4 (hard cover : alk. paper) ISBN-10: 0-12-373657-9 (hard cover : alk. paper) 1. Longevity–Physiological aspects. 2. Aging–Physiological aspects. 3. Inflammation. 4. Nutrition. I. Title. QP85.F466 2007 612.6′8–dc22 2007004576 British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. ISBN: 978-0-12-373657-4 For information on all Academic Press publications visit our Web site at www.books.elsevier.com Printed in the United States of America 07 08 09 10 9 8 7 6 5 4 3 2 1
  • v Preface xi Acknowledgments xiii Chapter 1 Inflammation and Oxidation in Aging and Chronic Diseases PART I 1.1. Overview 2 1.2. Experimental Models for Aging 10 1.2.1. Mortality Rate Accelerations 10 1.2.2. Mammals 12 1.2.3. Cultured Cell Models and Replicative Senescence 31 1.2.4. Invertebrate Models 32 1.2.5. Yeast 34 1.2.6. The Biochemistry of Aging 35 1.2.7. Biomarkers of Aging and Mortality Risk Markers 42 1.2.8. Evolutionary Theories of Aging 44 1.3. Outline of Inflammation 49 1.3.1. Innate Defense Mechanisms 50 1.3.2. Genetic Variations of Inflammatory Responses 54 1.3.3. Inflammation and Energy 56 1.3.4. Amyloids and Inflammation 59 1.4. Bystander Damage and Dependent Variables in Senescence 60 1.4.1. Free Radical Bystander Damage (Type 1) 61 1.4.2. Glyco-oxidation (Type 2) 63 1.4.3. Chronic Proliferation (Type 3) 63 1.4.4. Mechanical Bystander Effects (Type 4) 64 PART II 1.5. Arterial Aging and Atherosclerosis 65 1.5.1. Overview and Ontogeny 66 CONTENTS
  • 1.5.2. Hazards of Hypertension 74 1.5.3. Mechanisms 75 1.5.3.1. Inflammation 75 1.5.3.2. Hemodynamics 79 1.5.3.3. Aging 81 1.5.3.4. Endothelial Progenitor Cells 84 1.5.4. Blood Risk Factors for Vascular Disease and Overlap with Acute Phase Responses 84 1.6. Alzheimer Disease and Vascular-related Dementias 86 1.6.1. Neuropathology of Alzheimer Disease 87 1.6.2. Inflammation in Alzheimer Disease 91 1.6.3. Prodromal Stages of Alzheimer Disease 94 1.6.4. Overlap of Alzheimer and Cerebrovascular Changes 95 1.6.5. Insulin and IGF-1 in Vascular Disease and Alzheimer Disease 99 1.6.6. Blood Inflammatory Proteins: Markers for Disease or Aging, or Both? 101 1.7. Inflammation in Obesity 103 1.8. Processes of Normal Aging in the Absence of Specific Diseases 106 1.8.1. Brain 107 1.8.2. Generalized Inflammatory Changes in Normal Tissue Aging 107 1.9. Summary 112 Chapter 2 Infections, Inflammogens, and Drugs 2.1. Introduction 114 2.2. Vascular Disease 114 2.2.1. Historical Associations of Infections and Vascular Mortality 114 2.2.2. Modern Serologic Associations 115 2.3. Infections from the Central Tube: Metchnikoff Revisited 121 2.3.1. Humans: Leakage from Periodontal Disease and Possibly the Lower Intestine 121 2.3.2. Worms and Flies as Models for Human Intestinal Microbial Intrusion 125 2.4. Aerosols and Dietary Inflammogens 126 2.4.1. Aerosols 127 2.4.2. Food 129 vi Contents
  • 2.5. Infections, Inflammation, and Life Span 131 2.5.1. Historical Human Populations 131 2.5.2. Longer Rodent Life Spans with Improved Husbandry 136 2.6. Are Infections a Cause of Obesity? 142 2.7. Inflammation, Dementia, and Cognitive Decline 143 2.7.1. Alzheimer Disease 143 2.7.2. HIV, Dementia, and Amyloid 145 2.7.3. Peripheral Amyloids 147 2.7.4. Inflammation and Cognitive Decline During ‘Usual’ Aging 147 2.8. Immunosenescence and Stem Cells 150 2.8.1. Immunosenescence and Cumulative Exposure 150 2.8.2. Immunosenescence and Telomere Loss 152 2.8.3. Inflammation and Stem Cells 153 2.9. Cancer, Infection, and Inflammation 154 2.9.1. Helicobacter Pylori and Hepatitis B Virus 154 2.9.2. Smoking and Lung Cancer 156 2.10. Pharmacopleiotropies in Vascular Disease, Dementia, and Cancer 158 2.10.1. Anti-inflammatory and Anti-coagulant Drugs 158 2.10.2. Aspirin and Other NSAIDs 161 2.10.3. Statins 162 2.10.3.1. Vascular Disease 162 2.10.3.2. Dementia 164 2.10.4. Sex Steroid Replacement (Hormone Therapy) 165 2.10.5. Plant-derived Micronutrients and Neutriceuticals 169 2.11. Summary 172 Chapter 3 Energy Balance, Inflammation, and Aging 3.1. Introduction 176 3.2. Diet Restriction and Aging 177 3.2.1. Overview of Animal Models 177 3.2.2. Diet Restriction and Disease in Rodent Models 184 3.2.3. Diet Restriction, Starvation, Vascular Disease, and Longevity in Humans 186 3.2.4. Diet Restriction, Infections and Inflammation 192 3.2.5. Somatic Repair and Regeneration 199 Contents vii
  • 3.3. Energy Sensing in Diet Restriction and Satiety 200 3.3.1. Physiology 201 3.3.2. Biochemistry 202 3.3.3. Relevance to Arterial Disease and Cancer 210 3.4. Exercise, Cardiovascular Health, and Longevity 211 3.4.1. Humans 212 3.4.2. Rodent Models 215 3.4.3. Mechanisms in Exercise and Longevity 216 3.5. Diet, Exercise, and Neurodegeneration 219 3.5.1. Alzheimer Disease 219 3.5.2. Synaptic Atrophy in the Absence of Neurodegeneration 221 3.6. Laboratory Rodents as Models 225 for the ‘Couch Potato’ 3.7. Energy Balance in the Life History 228 3.8. Summary 231 Chapter 4 Nutrition and Infection in the Developmental Influences on Aging 4.1. Introduction 234 4.2. Synopsis of the Fetal Origins Theory 236 4.3. The Barker Studies of Infections 241 and Vascular Disease 4.4. Size, Health, and Longevity 245 4.4.1. Adult Height, Vascular Disease, and Longevity 246 4.4.2. Size at Birth and Adult Height 249 4.4.3. Criteria for Growth Retardation 252 4.4.4. Maternal Metabolism and Fetal Growth 254 4.4.5. Birth Size and Adult Vascular and Metabolic Disease 258 4.4.6. Twins: Small Size at Birth and Catch-up Growth, but Normal Longevity 262 4.5. Infection and Undernutrition on Birthweight and Later Disease 262 4.5.1. The Tangle 263 4.5.2. Maternal Infections and Nutrition 263 4.5.3. Smoking and Aerosols 267 viii Contents
  • 4.6. Infection and Nutrition in Postnatal Development and Later Disease 267 4.6.1. Diarrheas in Growth Retardation 267 4.6.2. Seasonal Effects 268 4.6.3. Serum Immune Response Markers of Chronic Infection in Health-Poor Children 270 4.6.4. Infections During Development 272 4.6.5. The Cost of Infections to Postnatal Growth: Evidence from Migration and Antibiotics 273 4.6.6. Unknowns 275 4.7. Famine 276 4.7.1. World War II (WWII) 276 4.7.2. 19th Century Famines 286 4.8. Maternal Physiology, Fetal Growth, and Later Chronic Disease 289 4.9. Growth in Adaptive Responses to the Environment 295 4.10. Genomics of Fetal Growth Regulation 298 4.10.1. Inherited Genetic Variations 299 4.10.2. Gene Imprinting: Inherited but Epigenetic Influences on Development 299 4.11. Summary 302 Chapter 5 Genetics 5.1. Introduction 306 5.2. Sources of Individual Variations in Aging and Life Span 306 5.3. Sex Differences in Longevity 310 5.4. Metabolism and Host-Defense in Worm and Fly 315 5.4.1. Metabolic Gene Signaling 315 5.4.2. Immunity and Metabolism 318 5.5. The Worm 323 5.5.1. Overview 323 5.5.2. Slower Eating Increases Life Span 325 5.5.3. Metabolism and Host Defense 325 5.6. Fly 329 5.6.1. Overview 329 5.6.2. Metabolism and Diet Restriction 330 Contents ix
  • 5.6.3. Heart 333 5.6.4. Infections, Host Defense, and Stress Resistance 335 5.6.5. Natural Variations in Longevity Pathways 337 5.7. Mammals 338 5.7.1. Growth and Metabolism 338 5.7.1.1. Rodent Mutants with Altered Insulin Signaling and Fat Metabolism 343 5.7.1.2. Human Hereditary Variations in Metabolic Genes 351 5.7.1.3. Size and Longevity 354 5.7.1.4. The Insulin-Sensitivity Paradox 355 5.7.2. Inflammation 355 5.7.3. Lipoproteins and Cholesterol Metabolism 357 5.7.4. ApoE4 Interactions with Diet, Cognition, and Vascular Aging 363 5.7.5. ApoE Alleles, Infection, and Reproduction 368 5.8. Summary 370 Chapter 6 The Human Life Span: Present, Past, and Future 6.1. Introduction 373 6.2. From Great Ape to Human 376 6.2.1. Human Life History Evolution 376 6.2.2. Chimpanzee Aging 383 6.2.3. The Evolution of Meat-Eating 385 6.2.4. Meat Adaptive Genes 391 6.2.5. The Increase in Life Expectancy 402 6.3. Four Major Shifts in Human Life History from Genetic and Cultural Evolution 404 6.4. The Instability of Life Spans 406 6.4.1. Infections 406 6.4.2. Air Quality 409 6.4.3. Obesity and Diabetes 410 6.4.4. Prospects 412 6.5. Summary of Chapters 1–6: Mechanisms in Aging and Life History Evolution 413 References 417 Name Index 599 Subject Index 625 x Contents
  • PREFACE xi Aging is a great scientific mystery. For 4 decades, I have been fascinated by the possibility of a general theory addressing genomic mechanisms in the continuum of development and aging in health and disease. While a Yale undergraduate in Biophysics, I was fortunate to be mentored by Carl Woese, who suggested that if I wanted to tackle a really new problem in little-trodden scientific territory, I should think about aging: “It is even more mysterious than development.” About 5 years later as a graduate student at “the Rockefeller,” I began work on neuroendocrine aspects of aging guided by Alfred Mirsky (McEwen, 1992). Mirsky was a major conceptualizer of differential gene expression in cell differ- entiation and development, including postnatal growth and maturation. Eric Davidson and Bruce McEwen, prior Mirsky students, were also key debaters in developing my thoughts on aging. In writing my PhD thesis, I tried to read everything published on biological and medical aspects of aging up to 1969. I chanced across two remarkable arti- cles by Hardin B. Jones (Jones, 1956, 1959). These papers, rarely cited at that time or since, showed the importance of cohort analyses to understanding aging. James Tanner also noted cohort effects in growth and puberty during the last 150 years (Tanner, 1962). Some readers of my thesis thought my attentions had strayed from my experiments by the emphasis I gave them: Tanner suspects that puberty occurs earlier because of decreased exposure to disease in childhood. Jones analyses has actually shown that the mor- tality of cohorts as children can be used to predict the mortality of these same cohorts as adults. If both conclusions prove true, there may be a com- mon site of action of the environment on the organ systems governing the length of mature life. (Finch, 1969, p. 11.) I was also was fortunate to learn some pathology as a graduate student at the Rockefeller by two masters of “in-the-gross” necropsy, Robert Leader and John Nelson, who taught me first-hand to use tweezers and scalpel and to see clues to pathology from the texture and color of tissues and fluids. “Old” John Nelson’s vast experience in rodent pathology helped me understand McCay’s observations that caloric restriction suppressed chronic lung disease (Chapter 3). Peyton Rous made a chilling comment after my thesis lecture (to the effect of): “Finch, I don’t see why you are wasting your time on a subject like aging—everyone knows aging is only about vascular disease and cancer.” Rous may yet be proved right, but to no chagrin in view of the thriving subject that has emerged and that may give a broad understanding of shared processes in many aspects of aging.
  • During the past 35 years, my research has remained focused on brain mech- anisms in aging. The turn toward inflammation began with molecular studies of Alzheimer disease about 15 years ago. My lab and others discovered that inflam- matory mechanisms were activated in Alzheimer disease (AD). Moreover, we showed that some glial inflammatory changes in AD also occur to lesser degrees during normal aging and can be detected before midlife. Furthermore, caloric restriction, which increases rodent life span, also retarded brain inflammatory changes. During this same decade, it became clear that vascular disease also involves slow inflammatory processes and that anti-inflammatory drugs reduce the risk of heart disease and possibly of Alzheimer disease as well. In the last 5 years, I have developed a major collaboration with Eileen Crimmins, a USC demographer whose work also showed the importance of inflammation in human health. Our papers address the questions of 4 decades back and have given the rationale for a new set of animal models being developed in collabo- ration with my close colleagues Todd Morgan and Valter Longo. Inflammation– diet interactions could explain the recent evolution of human longevity with caveats of its future potential. My inquiry necessarily leads to a broad range of evidence usually not consid- ered “on the same page” by highly focused researchers of specific diseases. The examples illustrate key points and cannot be comprehensive. I will try to indi- cate the level of certainty in evidence being considered and not try to explain “too much.” xii Preface
  • xiii I am grateful for the detailed comments and information given by Dawn Alley (NIA), Steve Austad (U Texas, San Antonio), David Barker (Oregon State U, MRC Southampton), Andrej Bartke (Southern Illinois U), Barry Bogin (U Michigan, Dearborn), Eileen Crimmins (USC), Greg Drevenstedt (USC), Rita Effros (UCLA), Doris Finch (Altadena, CA), Luigi Fontana (Washington U), Roger Gosden (Weill-Cornell Medical College), Michael Gurven (UC Santa Barbara), Shiro Horiuchi (Rockefeller U), Tom Johnson (U Colorado, Boulder), Marja Jylhä (U Tampere), Hillard Kaplan (U New Mexico), Edward Lakatta (NIA), Gary Landis (USC), Valter Longo (USC), George Martin (U Washington), Christopher Martyn (Winchester, UK), Edward Masoro (U Texas San Antonio), Roger McCarter (Penn State), Richard Miller (U Michigan, Ann Arbor), Charles Mobbs (Mount Sinai, NYC), Vincent Monnier (Case Western Reserve), Todd Morgan (USC), Wulf Palinski (U California, La Jolla), Kari Pitkänen (U Helsinki), Scott Pletcher (Baylor), Leena Räsänen (U Helsinki), Karri Silventoinin (U Helsinki), Craig Stanford (USC), Aryeh Stein (Emory U), John Tower (USC), and Paulus van Noord (Utrecht). And I am especially grateful to Eileen Crimmins and Valter Longo, who read all the chapters. Expert editorial assistance was provided by Jacqueline Lentz (USC), Bernard Steinman (USC), and Swamini Wakkar (USC); Bernard also masterfully developed the figures. The research from my lab was supported by the National Institute on Aging, the Alzheimer’s Association, the John Douglas French Foundation for Alzheimer disease, the ARCO/William F. Kieschnick Chair in Neurobiology of Aging, the Ellison Foundation for Medical Research, and the Ruth Ziegler Fund. Lastly and firstly, I could not have completed this project without the unvarying support of Doris Finch, who was always ready to relight the scholar’s lamp at flagging moments. ACKNOWLEDGMENTS
  • CHAPTER 1 Inflammation and Oxidation in Aging and Chronic Diseases PART I 1.1. Overview 2 1.2. Experimental Models for Aging 10 1.2.1. Mortality Rate Accelerations 10 1.2.2. Mammals 12 1.2.3. Cultured Cell Models and Replicative Senescence 31 1.2.4. Invertebrate Models 32 1.2.5. Yeast 34 1.2.6. The Biochemistry of Aging 35 1.2.7. Biomarkers of Aging and Mortality Risk Markers 42 1.2.8. Evolutionary Theories of Aging 44 1.3. Outline of Inflammation 49 1.3.1. Innate Defense Mechanisms 50 1.3.2. Genetic Variations of Inflammatory Responses 54 1.3.3. Inflammation and Energy 56 1.3.4. Amyloids and Inflammation 59 1.4. Bystander Damage and Dependent Variables in Senescence 60 1.4.1. Free Radical Bystander Damage (Type 1) 61 1.4.2. Glyco-oxidation (Type 2) 63 1.4.3. Chronic Proliferation (Type 3) 63 1.4.4. Mechanical Bystander Effects (Type 4) 64 PART II 1.5. Arterial Aging and Atherosclerosis 65 1.5.1. Overview and Ontogeny 66 1.5.2. Hazards of Hypertension 74 1.5.3. Mechanisms 75 1.5.3.1. Inflammation 75 1.5.3.2. Hemodynamics 79 1
  • 1.5.3.3. Aging 81 1.5.3.4. Endothelial Progenitor Cells 84 1.5.4. Blood Risk Factors for Vascular Disease and Overlap with Acute Phase Responses 84 1.6. Alzheimer Disease and Vascular-related Dementias 86 1.6.1. Neuropathology of Alzheimer Disease 87 1.6.2. Inflammation in Alzheimer Disease 91 1.6.3. Prodromal Stages of Alzheimer Disease 94 1.6.4. Overlap of Alzheimer and Cerebrovascular Changes 95 1.6.5. Insulin and IGF-1 in Vascular Disease and Alzheimer Disease 99 1.6.6. Blood Inflammatory Proteins: Markers for Disease or Aging, or Both? 101 1.7. Inflammation in Obesity 103 1.8. Processes of Normal Aging in the Absence of Specific Diseases 106 1.8.1. Brain 107 1.8.2. Generalized Inflammatory Changes in Normal Tissue Aging 107 1.9. Summary 112 2 The Biology of Human Longevity PART I 1.1. OVERVIEW Human life spans may have evolved in two stages (Fig.1.1A). In the distant past, the life expectancy doubled from the 20 years of the great ape-human ancestor during the evolution of Homo sapiens to about 40 years. Then, since the 18th cen- tury, life expectancy has doubled again to 80 years in health-rich modern popu- lations, with major increases in the post-reproductive ages (Fig. 1.1B) and decreases in early mortality (Fig. 1.1C). During these huge demographic shifts, human ancestors made two other major transitions. The diet changed from the plant-based diets of great apes to the high-level meat-eating and omnivory that characterizes humans. Moreover, exposure to infections increased. The great apes abandon their night nests each day and rarely congregate closely for very long in large groups. As group density and sedentism increased in our ancestors, so would their burden of infections and inflammation have increased from expo- sure to pathogens in raw animal tissues and from human excreta. I propose that the growth of meat-eating and sedentism selected for gene vari- ants adaptive in host defense and adaptive for high fat intake. Some of these genes may have favored the increased survival to later ages that enables the
  • Inflammation and Oxidation in Aging and Chronic Diseases 3 30 40 50 60 70 80 1700 1800 1850 1900 1950 20001600 2050 0 10 20 Phase 4 ? A 8 6 4 2 1 0.5 0.1 Shared ape ancestor 10 20 30 40 50,000 Million years ago Years before present 10,000 1,000 500 Homo sapiens ? ? ? ? ? Regenerative medicine Foragers Global Phase 3 Immunization/ antibiotics Y England Other Phase 1 Early urban Phase 2 Sanitation-nutrition Industrial Revolution ? Life expectancy, Y ? 10 100 90 80 70 60 50 40 30 20 0 0 10 9080706050403020 100 Y B 1751 1990 1870 0 10 9080706050403020 100 Y C 1.00000 0.00001 0.00010 0.00100 0.01000 0.10000 1751 1990 1870 Sweden: Survivors (%) Sweden: Cohort mortality rate FIGURE 1.1 Evolution of the human life span. A. Life expectancy from 6 million years ago (MYA) to present. Left panel is simplified from Fig. 6.1. The shared ancestor of chimpanzee and human is pre- dicted to have had a life expectancy at birth (qo) of 10 to 20 y, approximating that of wild chimps (Section 6.2.1). The range of q0 from 30 to 40 y in early, but anatomically modern H. sapiens is hypoth- esized here to approximate that of current human foragers (Gurven and Kaplan, 2007) (Section 6.2.1; Fig. 6.1, legend) and pre-industrial Europe, e.g., England (Right panel). Life expectancy may have increased during the increase of brain size after 1.8 MYA (see Fig. 6.1 legend). Early Homo as a species was established by 1.8 MYA (Section 6.2) (Right panel). The major increase of life span speculatively began during later stages in evolution of H. sapiens, 0.5 to 0.195 MYA (see Fig. 6.1 legend and Section 6.2.2). Right panel, adapted from Oeppen and Vaupel (2002), Suppl. Fig. 5 and framed by historical markers of my interpretation. Data for England 1571–1847 from op. cit.; mean 36.2 y [30.6-41.7 y, 95% CI] calculated from Paine and Boldsen (2006), p.352 and (Wrigley and Schofeld, 1997). Global average life expectancy at birth in 2006: 64.8 y (weighted average, The World Factbook (CIA, 2006). B. Survival curves for Sweden showing the progressive increase in life span and rectangularization of the survival curves from 1751 to present. From Human Mortality Database. C. Mortality rate curves and aging (semi-log scale) for Sweden 1751, 1870, 1990, showing the historical trends for progressive downward shift of the entire mortality curve (See Fig. 2.7).
  • uniquely human multi-generational caregiving and mentoring. Many such genetic changes had probably evolved by the time of the Venus of Willendorf (cover pho- tograph), 21,000 years ago in the Upper Paleolithic. Her manifest obesity may be viewed as adaptive in times of fluctuating food, with few ill consequences during the short lifespans of the pre-modern era, at the least, fewer than in the modern era of rampant chronic obesity. However, the most recent and rapid increases in life span cannot be due to the natural selection of genes for greater longevity. I emphasize the plural lifespans, because many concurrent human life history schedules can be recognized in the world today that differ by the rate of growth, age of puberty and sexual maturation, the schedule of reproduction, and life expectancy. Evolutionary biologists recognize the huge plasticity of life history schedules, which vary between populations and respond rapidly to natural or artificial selection (Section 1.2.8). In the not too distant past, human life histories and lifespans may have been outcomes of natural selection, whereas changes in the last 200 years are clearly driven by culture and technology. I propose that the evolution of the human life span depended on the genetic modulation of synergies between inflammation and nutrition. These dyadic syner- gies are both substrates and drivers of specific chronic diseases and dysfunctions (Fig. 1.2A). Many aspects of aging are accelerated by infections and inflammation, while drugs and nutritional interventions that slow aging may act by attenuating inflammation and oxidative damage. The current lab models selected for fecundity in atypically clean environments with unlimited food and no stress from predators may not represent aging processes in the bloody, dirty, invasive, and stingy envi- ronment of natural selection. Host defense and somatic repair processes are evolved to survive the relentless assaults by microorganisms, parasites, and other predators that are omnipresent in the natural environment.Understanding gene-environment (G x E) interactions in the inflammation-nutrition synergies is fundamental to human aging, past, present, and future. No single gene or mechanism is likely to explain human aging and its evolution, because natural selection acts mainly through successions of small quantitative gene effects. Many gene variants show trade-offs in balancing selection, epitomized by the sickle-cell gene in resistance. A broad theory of aging may emerge by mapping the nutrition-inflammation syn- ergies of pathological aging changes (Fig. 1.2) and their role in oxidative damage. Because host defense and repair require energy, homeostatic energy allocation strategies were evolved for eco-specific contingencies. High infectious burdens and poor nutrition attenuate somatic repair and growth (Fig. 1.2B). Homeostatic resource allocation involves insulin-like metabolic pathways that operate throughout devel- opment and adult life (Fig. 1.3). Insulin-like signaling pathways were recently shown to influence aging in many species. Many aspects of aging at the molecular and cell level can be attributed to ‘bystander’ damage from locally generated free radicals in the immediate microenvironment. DNA, lipids, and proteins are vulnerable to bystander oxidative damage from ROS produced by activated macrophages and from spontaneous reactions with glucose and other sugars. In turn, oxidatively damaged molecules interact with, and can stimulate, inflammatory processes. 4 The Biology of Human Longevity
  • FIGURE 1.2 A. Pathways linking infection and inflammation in aging. Adapted from (Crimmins and Finch, 2006a); drawn by Aaron Hagedorn (USC). B. Energy allocation pyramid in health and during infection, showing energy reallocations during infections, which may cause acute and chronic energy deficits. Human basal metabolism is increased 30% by systemic infections (sepsis) and 15% by sickle cell disease (Lochmiller, 2000). The acute phase inflammatory responses decrease appetite and induce lethargy (sickness behavior). Fever burns energy and increases basal metabolism 25–100% (Roe and Kinney, 1965; Waterlow, 1984): For each 1˚C of temperature elevation during fever, human basal metabolism is increased by 10–15%. It is unknown how much energy is consumed by immune cell proliferation and the increased production of CRP and other acute phase proteins. The major realloca- tion of energy during inflammation comes at the expense of voluntary activity and growth (Chapter 4). Pathways linking infection and inflammation to mortality ExternalInternal Diet Drugs Infection Noninfectious Inflammogens Dyslipidemia A Inflammation Growth and Development Atherosclerosis & Thrombosis Other Organ Damage Morbidity & Mortality T cell activation Infections Depletion Resource allocation Reproduction - growth - repair Locomotion-behavior Host defense, acute phase Basal metabolism Healthy B Infected Inflammation and Oxidation in Aging and Chronic Diseases 5
  • 6 The Biology of Human Longevity This analysis of the complex interactions in the aging of humans and animal models is guided by three “Queries” about inflammation, nutrition, and oxidant stress during aging. (QI) Does bystander damage from oxidative stress stimulate inflammatory processes? (QII) Does inflammation cause bystander damage? (QIII) Does nutrition influence bystander damage? These Queries are not posed as testable hypotheses because in each domain, multiple outcomes are expected from the trade-offs present throughout natural selection. Consequently, many exceptions are expected in the direction and degree of these associations. The evidence shows that inflammatory and oxidant damage accumulated by long-lived molecules and cells promote the major dysfunctions of aging that, in turn, drive the acceleration of mortality during aging. Later life dysfunctions of the vasculature, brain, and cell growth may be traced to prodromal (subclinical) inflammatory changes from early in life. These processes are examined across the stages of life history, from oogenesis, fetal and postnatal development, and adult stages into senescence. This inquiry considers aging as a process that is event-related, rather than time-related, from fertilization to later ages (Finch, 1988; Finch, 1990, p.6). Degenerative changes that eventually lead to increased mortality risk can be ana- lyzed as bystander events from agents acting ‘without and within.’ External agents include infections and physical trauma. Internal agents include free radi- cals produced by macrophages in host defense and subcellularly by mitochon- dria through normal metabolism. Most long-lived molecules inevitably accumulate oxidant damage during aging. Arterial aging demonstrates many bystander processes which stimulate inflammatory pleiotropies (multiple targets of a process) and which are major risk factors for mortality from heart attack and stroke. Diabetes and infections cause oxidative damage and accelerate arterial changes through complex recursive pathways (Fig. 1.2A). The theory of inflammation and oxidative stress in aging draws from the free radical immunological and inflammatory theories of aging and the Barker theory of fetal origins of adult disease (Chapter 4). Free-radical causes of cancer and of aging ‘itself’ were hypothesized by Denham Harman in 1956 to involve genetic damage (Harman, 1956, 2003). Then, in the next decade, Roy Walford’s immunological theory of aging extended the importance of somatic cell variation from mutations and other autogenous aging changes to autoimmune reactions, in which somatic cell neoantigens caused pathological aging (Walford, 1969). Since then, the free rad- ical hypothesis was extended to many aspects of aging through mechanisms that involve oxidant stress (Bokov et al, 2004; Harper et al, 2004; Schriner et al, 2005; Stadtman and Levine, 2003; Beckman and Ames, 1998). Damage from inflammation is now well recognized in aging processes and chronic diseases and is mediated by
  • Anti-inflammatory and Anti-atherogenic Pro-inflammatory and Pro-atherogenic IGF-1 PI3-K Akt NO B NOS Cardiomyocyte and stem cell proliferation Ang II Oxidized LDL TNFα Vascular endothelial apoptosis Platelet aggregation ROS Scavenging Vasodilation FIGURE 1.3 For legend see page 8. Inflammation and Oxidation in Aging and Chronic Diseases 7 Ligands Yeast Worms Files Mico Humans Growth hormone IGF-1IGF-1 IGF-1−receptorIGF-1−receptor RasRas Growth hormone Glucose Gpr1 Ras2 Cyr1 (cAMP) PKA Msn2, Msn4 SOD, cotainse, haps, glycogen accumulation, ...? (Growth) (Growth) DAF-16 SOD, catalase, Haps, fat and glycogen accumulation, ...? SOD, fat accumulation, ...? SOD, catalase, Hsps, fat accumulation, ...? (Growth) (Growth) (Growth) fat accumulation Akt/PKBAkt/PKBAkt/PKBAkt/PKB DAF-2 Insulin/IGF-1-like Insulin/IGF-1-like INR CHICO P13K (Ptdins-3-Ps)P13K (Ptdins-3-Ps)P13K (Ptdins-3-Ps)AGE-1 (Ptdins-3-Ps) ? ? ? ? ? ? ? ?? ? Aging Aging Aging Aging/diseases ? ? Sch9(45-47% identical to Act PKA) A Receptors G-proteins Second messengers Serinel Threonine-kineses Stress resistance transcription factors Stress-resistance proteins
  • free radicals and many specific inflammatory peptides (Beckman and Ames, 1998; Ershler and Keller, 2000; Finch and Longo, 2001; Finch, 2005; Franceschi et al, 2005; Wilson et al., 2002). Inflammation was already recognized in arterial disease a century ago by Rudolf Virchow (Bokov et al, 2004) (Section 1.5.3). One aspect of immunosenescence in Walford’s theory has been recognized as the depletion of naive T-cells and the acquisition of memory T-cells that are present in unstable arterial plaques. Most recently, Barker’s ‘fetal origins of adult disease’ identifies maternal nutritional influences on adult vascular and metabolic diseases (Chapter 4). I will argue that exposure to infection and inflammation during development also have major importance to outcomes of aging. While the ‘aging’ risk factor in the chronic diseases is well recognized demo- graphically, aging changes are often neglected in the disease mechanisms. There are many disconnects between the field of ‘basic’ aging and the biomedical fields of chronic diseases (Alzheimer, cancer, diabetes, vascular disease, etc.). I argue most age-associated diseases interact throughout with ‘normal aging processes.’ Many of the same molecules, cells, and gene systems that are altered during aging are considered separately by research in Alzheimer, cancer, and arterial diseases. Major shared mechanisms in aging and disease may be found to stem from roots in the common soil of aging. Shared mechanisms in aging are emerging in the genet- ics of aging, the insulin-like signaling pathways of metabolism in yeast, flies, worms, and mammals that influence longevity (Fig. 1.3A). Insulin signaling also operates in human arterial disease (Fig. 1.3B). These convergences of aging processes imply ancient genomic universals in life span evolution. It is time to reach for a more gen- eral theory that encompasses ‘normal aging’ and ‘diseases of aging’ in the context of evolution and development. However, we should not expect that gene regula- tion of longevity and senescence will operate by the strict gene regulatory circuits that govern early development (Davidson, 2006; Howard and Davidson, 2004). 8 The Biology of Human Longevity FIGURE 1.3 Insulin-like metabolic signaling pathways in longevity and vascular disease. A. Yeast, worms, flies, and mice share metabolic pathways with conserved elements that modulate life span. From (Longo and Finch, 2003). B. Insulin/IGF-1 pathways in vascular disease. Redrawn and adapted from (Conti et al., 2004). IGF-1 strongly promotes the survival of vascular smooth muscle cells, whereas Low plasma IGF-1 is associated with many cardiovascular risk factors (Section 1.6.5). This sketch suggests cardioprotective (positive) and atherogenic-inflammatory (negative) limbs of the response (left and right sides) (Conti et al., 2004; Che et al., 2002). Positive: IGF-1 binding increases nitric oxide production via a PI3K-Akt cascade, which increases vasodilation and ROS scavenging, while inhibiting platelet aggregation and endothelial apoptosis (Isenovic et al. 2002, 2004; Conti et al., 2004; Dasu et al., 2003). Additionally, activation of IGF-1 receptor and Akt stimulates the proliferation of cardiomyocytes and stem cells (Linke et al., 2005; Catalucci and Condorelli, 2006). Negative: Oxidized LDL directly inhibits IGF-1 receptor levels in vascular smooth muscle cells (Scheidegger et al., 2000). However, TNF␣ may synergize with IGF-1 receptor through the Gab1 subunit to enhance adhesion and other inflammatory proatherogenic activities (Che et al., 2002).
  • Next is an overview of this book. Chapter 1 has two parts: Part 1 reviews human aging and age-related diseases for a diverse readership, emphasizing inflamma- tion and major experimental models. An overview of inflammation and oxidant stress in host defense suggests a classification of bystander damage. Mechanisms of inflammation are outlined, including the energy costs. Part 2 reviews in more detail inflammation in arterial and Alzheimer disease. These details are critical to understanding human aging and the role of insulin-like metabolic pathways. Many inflammatory processes emerge during ‘normal’ or usual aging, but in the absence of specific diseases. The slow creep of inflammation from early years may drive the accelerating incidence of chronic diseases. This hypothesis is supported by evidence that many diseases benefit from drugs with anti-inflammatory and anti-coagulant activities (Chapter 2) and by energy (diet) restriction, which can have anti-inflammatory effects (Chapter 3). Chapter 2 examines environmental inflammatory factors in vascular disease and dementia with a focus on infections, environmental inflammogens, and drugs that modulate both vascular disease and dementia. Infections and blood levels of inflammatory proteins are risk factors for future coronary events and possibly for dementia. When early age mortality is high, the survivors carry long- term infections that impair growth and accelerate mortality at later ages (‘cohort morbidity phenotype’). Chronic infections, which are endured by most of the world’s human and animal populations, cause energy reallocation for host defense. Infections and inflammation may impair stem cell generation, with consequences to arterial and brain aging. Diet may introduce glycotoxins that stimulate inflammation. Some anti-inflammatory and anti-coagulant drugs may protect against coronary artery disease and certain cancers, and possibly also for Alzheimer disease. These ‘pharmacopleiotropies’ implicate shared mechanisms in diverse diseases of aging. Energy balance, inflammation, and exercise are addressed in Chapter 3. Diet restriction, which can slow aging and increase life span, also alters insulin-like signaling. Moreover, diet restriction attenuates vascular disease and Alzheimer disease in animal models, again suggesting common pathways. Diet restriction in some conditions has anti-inflammatory effects and may attenuate infections. Conversely, hyperglycemia is proinflammatory in obesity and diabetes. Exercise and energy balance influence molecular and cellular repair, in accord with evolutionary principles. Chapter 4 considers developmental influences of infections, inflammation, and nutrition on aging and adult diseases. Birth size, overly small or excessively large, can adversely affect later health through complex pathways. Developmental influences attributed to maternal malnutrition in the Barker hypothesis are extended here to infections. Fogel’s emphasis on malnutrition as a factor in poor health can also be extended to include consequences of infection and inflamma- tion. I argue that infection and inflammation compromise fetal development by diverting maternal nutrients to host defense, with consequences to development that influence adult health and longevity. Inflammation and Oxidation in Aging and Chronic Diseases 9
  • Chapter 5 reviews genetic influences on inflammation, metabolism, and longevity in animal models and humans. Mutations in insulin-like metabolic path- ways shared broadly by eukaryotes can also influence longevity. These metabolic pathways (Fig.1.3A) also interface with inflammation. Certain mutations of insulin signaling that increase the worm life span also increase resistance to infections. The human apoE alleles influence many aspects of aging and disease; the apoE4 allele shows population differences in frequency and effects that may prove to be exemplars of gene-environment interactions during aging. The last chapter considers the evolution of human life span from shorter-lived great ape ancestors that ate much less meat and lived in low density populations. Human longevity may have evolved through ‘meat-adaptive genes’ that allowed major increases of animal fat consumption and increased exposure to infection and inflammation not experienced by the great apes. The book closes by dis- cussing environmental trends and obesity, which may influence future longevity. 1.2. EXPERIMENTAL MODELS FOR AGING Aging and senescence in yeast, fly, worm, rodent, monkey, and human are reviewed with details referred to in later chapters. Lab models are referred to by their common names: fly (Drosophila melanogaster); monkey (rhesus, Macaca mulatta); mouse (Mus musculus); rat (Rattus norvegicus); worm (roundworm, nematode Caenorhabditis elegans); yeast (baker’s yeast, Saccharomyces cerevisiae). Other related species may have different life histories (Finch, 1990) and are identified by full name where discussed. At the population level, humans and these models share the characteristics of finite life spans determined by accelerating mortality. These species share the characteristic of female reproductive decline and oxidant damage in many cells and tissues during aging. Each species has a canonical pattern of aging that per- sists in diverse environments (Table 1.1) (Finch, 1990). Insulin-like metabolic sig- naling influences life span, as shown by mutants (Fig. 1.3A) (Chapter 5), suggesting a core of shared mechanisms in aging. However, lab flies and worms differ importantly from mammals by the absence of tumors during aging. The recent discovery that the adult fly gut has replicating stem cells that replace the epithelium with 1 week turnover (Ohlstein and Spradling, 2006) could give a basis for tumor formation in longer-lived species, such as honeybee queens. 1.2.1. Mortality Rate Accelerations All individual organisms have finite life spans, it is simple to say. The core issue in aging is to resolve environmental effects on endogenous aging processes. The hugely complex gene x environment interactions collectively result in mortality risks that define the statistical life span. Here, we face the immense challenge of moving the level of causal analysis from populations to the indi- 10 The Biology of Human Longevity
  • vidual. Time (age) is the best predictor of future longevity in populations. However, the multifarious aging changes that can be identified in individuals are much weaker predictors of longevity risk, the elusive ‘biomarkers of aging’ discussed below. Senescence in populations of humans and many other species can be com- pared by the rate of mortality acceleration during aging (Fig. 1.1C) (Finch, 1990, pp. 13–16; Finch et al, 1990; Johnson et al, 2001; Nusbaum et al, 1996; Pletcher et al, 2000; Sacher, 1977). In humans and rodents, mortality accelerations arise soon after puberty (Fig. 1.1C). The lowest values of mortality, which occur in mammals at about puberty, are designated as initial mortality rates (IMRs) (Finch et al., 1990; Finch, 1990, pp. 13–16). The main phase of mortality acceleration is described by the exponential coefficient of the Gompertz equation (Table 1.2). In humans, flies, and worms, mortality rates decelerate at later ages (Finch, 1990, p. 15) and (Carey et al, 1992; Johnson et al, 2001; Vaupel et al, 1998). Mortality deceleration at later ages is less definitive in lab rodents (Finch and Pike, 1996). These complex curves may also be fitted by multi-stage Gompertz (Johnson et al, 2001) or Weibull equa- tions (Pletcher, 2000; Ricklefs and Scheuerlein, 2002). The mortality acceleration in both equations is the strongest determinant of life span in most populations. The Gompertz exponential coefficient is conveniently expressed as the ‘mor- tality rate doubling time’ (MRDT), which ranges 1000-fold between yeast and long-lived mammals (Table 1.2) (Finch, 1990, pp. 662–666). Yeast, worm, and fly show the most rapid senescence, while birds and mammals show gradual senes- Inflammation and Oxidation in Aging and Chronic Diseases 11 TABLE 1.1 General Characteristics of Aging (Canonical Patterns of Aging) Mortality Reproductive Slowed Cardiac/Vascular Abnormal Oxidative Brain Neuron Accelerationa Declineb Movementc Dysfunctionsd Growthse Damagef Loss During Agingg yeast + + not relevant not relevant 0 + not relevant fly + + + + 0 + yes worm + + + not relevant 0 + not likely mouse, + + + + + + sporadic except rat in disease monkey + + + + + + “ human + + + + + + “ a. see Table 1.2 b. yeast, budding diminishes; fly and worm, egg production diminishes before death; mammalian ovary becomes depleted (Finch, 1990). c. spontanteous locomotion: fly (Finch, 1990, p. 65); worm, idem, p. 560; mammals (Slonaker, 1912) and common knowledge. d. fly, slowed pulse and lower threshold for fibrillation (Section 5.6.3, Fig. 5.7) and vascular changes in other insects ibid p.65; rodent, loss of arterial elasticity, myocardial fibrosis, and atheroma (Sections 1.2.2 and 2.5); monkey, coronary artery disease induced by fat (Clarkson, 1998); human, Sections 1.2.2 and 1.5, Fig. 1.4 and 1.6. e. fly and worm, no tumor observed in wild-type. The presence of dividing stem cells in the adult fly gut (Ohlstein and Spradling, 2006) might lead to tumors in long-lived fly species that over-winter. f. worm, fly, Section 1.2.4; rodent and human, Sections 1.2.4 and 1.6.2. g. Section 1.2.2.
  • cence. At the other extreme is the theoretical limit of ‘negligible senescence’, with MRDTs of >100 years (Finch, 1990, pp. 206–247; Finch, 1998; Vaupel et al., 2004). Species of long-lived fish (Cailliet et al, 2001; De Bruin et al, 2004; Geuerin, 2004), turtles (Congdon, 2003; Henry, 2003; Swartz, 2003), and conifers (Lanner and Connor, 2001) have not shown reproductive aging and are candidates for negligible senescence; however, data are lacking to evaluate mortality rates. MRTDs within a species vary less than the 10-fold or more variations in IMR. Human populations show a remarkable 10-fold range of IMR variations (Table 1.2), which reflect the level of health allowed by nutrition, infections, and other environmental factors (Chapters 2, 3, 4). Experimental variations of MRDT include 2-fold difference by diet (diet restriction in rodents, Chapter 3) and geno- type (Age-1 worm mutant, Chapter 5). Curiously, rodent MRTDs do not vary much by genotype, despite quite different diseases of aging (Finch and Pike, 1996). Human MRTDs are fairly similar across populations, despite major differ- ences in diseases and overall mortality (Finch, 1990; Gurven and Kaplan, 2007), e.g., Sweden (Table 1.2; Fig. 1.1C and Fig. 2.7). Male mortality is generally higher throughout life (Section 5.3). 1.2.2. Mammals Mammalian aging follows canonical patterns that gradually emerge after matura- tion and progress across the life span in proportion to the species life span (Finch, 1990). The seeds of aging are found before birth in many tissues, e.g., arteries and ovaries, as discussed below. The occurrence of these aging patterns in at least 5 of the 28 orders of placental mammals implies shared gene regula- tory systems evolved hundreds of million years ago that determine the level of 12 The Biology of Human Longevity TABLE 1.2 Comparative Demography of Aging Initial Mortality Rate (IMR) Mortality Rate Doubling Time (MRDT) Maximum Life Span yeasta 0.2/d 10 d >20 d flyb 0.1/d 5 d >60 d mouse, ratc 0.1/mo 4 m > 48 mo human Sweden)d 1751 0.0090/y 7–9 y <100 1931 0.0008/y >110 y These organisms show exponential accelerations of mortality, approximating a straight line on a semi-logarithmic plot of mortality rates against age (Fig. 1.1B), as described by the Gompertz equation for mortality rates: m(x) = Aexp(αx), where α is the Gompertz coeffi- cient, x is age, and A is the initial mortality rate, IMR. Mortality rate doubling time is calculated as ln 2/α (Finch et al, 1990). Rodents fed ad libitum. a, yeast (Finch, 1990, p. 105) from data of (Fabrizio et al, 2004), chronologic model (non-dividing); b, fly B stock (Nusbaum et al, 1996); c, representative rodent strains (Finch and Pike, 1996); d, Swedish historical populations (Finch and Crimmins, 2004, 2005; Crimmins and Finch, 2006a, b), and unpublished. IMR is calculated differently by species according to conventions. For rodents and human, IMR is calculated at the age of sexual maturation (puberty), its lowest value. For worm and fly, IMR is calculated at age 0 (hatching). Also see Table 5.1 and Finch (1990), pp. 663–666.
  • molecular and cell turnover and repair in specific tissues. The canonical patterns of aging thus can be considered as genetically programmed aging. The increasing incidence of diseases of aging corresponds to the acceleration of mortality during aging, as known in detail for humans and rodents. Arterial disease (heart attack, stroke) and cancer are the main causes of death across aging human populations (Fig. 1.4). Vascular deaths increase more or less expo- nentially after age 40, whereas breast cancer incidence plateaus after menopause. By age 65, vascular deaths exceed those from cancer in most populations (Horiuchi et al, 2003). In 1985, in Japan, Sweden, and the United States, for example, the total male deaths recorded for heart attack and stroke were 2-fold or more than for cancers, 3-fold more than respiratory conditions, and 30-fold more than for infectious diseases (Fig. 1.4) (Aronow, 2003; Himes, 1994; Horiuchi et al, 2003). The relative proportion of heart attacks (ischemic heart disease) and stroke (cerebrovascular disease) vary between populations. However, by 2002 in the United States, cancer mortality appears to have overtaken vascular-related mortality for age 85 and younger, where about 5% more died of cancer than from heart disease (476,009 vs. 450,637) (American Cancer Society, 2006). The cam- paigns on prevention and intervention of vascular disease are having remarkable impact on vascular changes. In rodents, the incidence of new pathologic lesions also increases exponen- tially (Bronson, 1990; Simms and Berg, 1957; Turturro et al, 2002), and roughly Inflammation and Oxidation in Aging and Chronic Diseases 13 0 10 20 30 40 50 60 Percent Japan Sweden U.S. Vascular Cancer Infections Causes of Death, Men (75+, Y) FIGURE 1.4 Circulatory diseases and cancer are the major cause of death in Japan, Sweden, and U.S. males aged 75+ in 1985. Circulatory diseases include ischemic heart disease and cerebrovascular disease. Very recently, cancer in the United States has risen to be the major cause of mortality before age 85, due to the remarkable success of the vascular disease campaigns. (Graphed from Table 4 of Himes, 1994).
  • 14 The Biology of Human Longevity paralleling the acceleration in mortality rates (Fig. 1.5). Diet restriction shifts the incidence of lesions to later ages and slows the acceleration of mortality (Chapter 3). The causes of death are often unresolvable, because multiple lesions are common at later ages (Fig. 1.5 and legend). The Berg-Simms colony founded in 1945 gives an unsurpassed documentation of age-related degenerative disease and mortality (Berg, 1976). Despite the relatively primitive husbandry and hygiene, life span was in the current range. The pathology of aging (specific organ lesions and age incidence) 100 200 3000 400 900 1000 +0.5 −1.0 −2.0 Age in Days LOGP Total of Fiv e Diseases Ch ronicNephrosis,etc. M yocardial Degen. Periart eritis Mortality MuscularDegen. 0.1 0.01 1.0 1.00.1 10.0 PDeath P Lesion Pit.Tum or FIGURE 1.5 The incidence of new pathologic lesions in rats increases exponentially in parallel with accelerating mortality. Similarly, in C57BL/6NNia mice the percentage of mice with more than three lesions doubled every 6 months: 12 m, 20.4%; 18 m, 41.7%; 24 m, 75.9% (Bronson, 1990). This doubling rate is slower than that of mortality rate doubling of 3.6 m, calculated from the Gompertz slope (Finch and Pike, 1996). Although the Berg-Simms colony was founded in 1945 before labora- tory animal infections were well controlled, their ‘rat palace’ at the College of Physicians and Surgeons (Columbia U) had little respiratory disease (<5% of rats). Rats were not selectively inbred, except to eliminate an ‘eye anomaly’ (Simms and Berg, 1957). Female life span: median 31 m, max- imum 34 m; male life span: median 27 m, maximum 29 m (Berg, 1976).This level of health and longevity is remarkable for that time. (Redrawn from Simms and Berg, 1957.)
  • Inflammation and Oxidation in Aging and Chronic Diseases 15 (Simms and Berg, 1957; Simms and Berg, 1962) has been confirmed in modern colonies (Bronson, 1990). Kidney lesions preceded tumors and cardiomyopathy; arterial calcification was occasional. In current colonies, kidney lesions and tumors also predominate, occurring in 80% of aging rodents across genotypes (Bronson, 1990; Turturro et al, 2002). Myocardial lesions are less common than in the Berg-Simms era and may vary; e.g., in aging C57BL/6 mice, myocardial degen- eration ranged from 8% (Bronson, 1990) to 40% (Turturro et al, 2002), always less than tumors and kidney lesions. The arterial and myocardial pathology in early colonies is discussed in Section 2.5.2, together with improvements in hygiene and husbandry that increased life span with some parallels to the recent human improvements. Rodents in modern colonies on standard diets are not thought to die from arterial degeneration or thrombosis. This may be incorrect. Rodent models for aging have been borrowed from existing lines that were originally developed for genetic studies of cancer and other chronic diseases, and of transplantation (immunogenetics). Rodents with delayed incidence of pathology until after 18 m were used as controls for early onset tumors, e.g., the relatively long-lived C57BL/6J and DBA/2J mice. All baseline stocks were selected for traits of fast growth and high fecundity, which is the rule for domestication for animals and plants. Infectious diseases were gradually minimized. The resulting models differ importantly from their feral origins, i.e., the true ‘wild-types.’ For example, wild-caught mice are smaller, mature later, and live longer than lab mice (Miller et al, 2002). Moreover, diet restric- tion has much less effect on life spans of wild-caught mice (Harper et al, 2006) (Chapter 3, Fig. 3.3). Immune functions also differ in ‘unhygienic’ feral mice and rats, with much higher levels of autoreactive IgG (Devalapalli et al, 2006). The modern lab rodents with unlimited access to food, low physical activity, and tendencies to obesity may thus be fine models for contemporary lifestyles. However, the limited exposure to infections is unlike the real world. It may be necessary to incorporate antigenic challenges in our aging animal colonies to understand the aging mechanisms at work in human populations, past, present, and future. In humans, arterial degenerative aging changes result from two long-term processes: the inexorable progressive accumulation of arterial wall lipids (Fig. 1.6A) and arterial rigidity, both from starting early in life (Sections 1.2.6 and 1.6). The loss of elasticity increases blood pressure (Fig. 1.6B), independent of clinical hypertension syndromes. The atherosclerotic lesions can lead to clots (thromboses) that block blood flow with catastrophic effects. Mortality from ischemic heart disease and stroke increases exponentially with adult age (Fig. 1.6C). Systolic pressure elevations are major risk factors in heart attack and stroke and are as universal to human aging as menopause and bone thinning. The loss of arterial elasticity and artery wall thickening (arteriosclerosis) are ubiq- uitous in mammals, while focal atherosclerosis is more prominent in humans and primates than rodents (Tables 1.1 and 1.4). The aorta and other central arteries become progressively thicker. The accumulation of oxidized lipids begins before birth
  • 16 The Biology of Human Longevity 0 A 100 600 500 400 300 200 0 10 30 807060504020 Y Abdominal Aorta Cumulativelesionarea(103µm2) 90 150 140 130 120 110 100 5 B 15 25 35 45 55 65 75 85 mmHg Systolic Pressure with Age Y Framingham (1997) US NHANES (1993) Nichols validation (1985) CTLDR CRON (2004) FIGURE 1.6 Arterial aging in humans. A. Arteries accumulate lipids progressively throughout life. The area of abdominal aorta surface covered by lipid-rich deposits (oil red O staining) increases progressively during postnatal life. (Redrawn from D’Armiento et al, 2001.) B. Increases of blood pressure with age (cuff pressure) are widely observed across human populations and are major risk factors in heart attacks and stroke. (Redrawn from O’Rourke and Nichols, 2005.) DR (diet resriction) and CTL (control) from CRON study of the Calorie Restriction Society (Fontana et al, 2004) (Section 3.2.3).
  • 1 2 4 8 16 32 64 128 256 120 C 180160140 1 2 4 8 16 32 64 128 256 120 180160140 Age at risk: 80-89 40-49 50-59 60-69 70-79 CADMortalityRisk Systolic pressure (mm Hg) Age at risk: 80-89 50-59 60-69 70-79 StrokeMortalityRisk Systolic pressure (mm Hg) FIGURE 1.6 (continued) C. Mortality from coronary artery disease (CAD) and stroke increases expo- nentially with elevated systolic pressure. Aging (40–89 years) increases the risk over a 50-fold range at each blood pressure level. There is no apparent threshold or cut-off for protection against adverse effects of blood pressure elevations. (Redrawn from Lewington et al, 2002.) D. Progressive increase in the glycation of human aortic elastin with aging. (Redrawn from Konova et al, 2004.) The chem- istry of these fluorescent products is uncharacterized. Elastin has a very long molecular life span in the adult aorta, as judged by the linear accumulation of racemized D-aspartate up through age 80 (“D-elastin”) (Powell et al, 1992). Collagen, however, continues to turn over, as indicated by the smaller increase of D-aspartate (“D-collagen”). 0 30 25 10 5 15 20 0.00 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 20 40 60 800 D D-elastinD-collagen D-elastin D-collagen AGE-elastin AGEelastin
  • 18 The Biology of Human Longevity in microscopic cell clusters (Section 1.5.1). The numerous inflammatory changes include increased macrophages, free radical producing enzymes (NADPH oxidase), cell adhesion molecules (ICAM), cytokines (TGF-β1), and matrix met- aloproteinases (MMP-2 and -9). These diffuse changes are generally independ- ent of focal atheromas. Thus, oxidative damage (oxidized lipids) and inflammation are at work from the beginning in arterial aging (Queries I and II). Arterial elasticity decreases progressively from alterations in collagen and elastin by inter-molecular AGE adducts (advanced glycation and glyco-oxidation end products) derived from glucose and other reducing sugars (Fig. 1.6D) (Section 1.4.4). AGE adducts contribute to arterial rigidity by intermolecular cross-links between collagen and other proteins. In turn, AGE may kindle local inflammation by activating scavenger receptors. Arterial elastin is very-long lived, as shown by accumulations of racemized D-aspartate (Fig. 1.6D). Racemization spontaneously converts normal L-amino acids to the D-isomers. In long-lived proteins, the accu- mulation of ‘racemers’ is a direct marker of age (Bada et al, 1974; Helfman and Bada, 1975). Because veins undergo less wall thickening, arterial aging is hypoth- esized to be driven by the repeated pressure waves at each pulse (Section 1.5.3.2). Blood flow patterns modify gene expression in atheroprone arterial regions. New macroscopic atheromas appear throughout life. Lipid oxidation may be a key cause of atheroma initiation and progression (Queries 1 and 2). Inflammatory processes are active throughout atherogenesis and are intensified in atheroprone arterial zones. The developing atheromas are described as a complex wounding response with cell growth and cell recruitment; oxidation of lipids and proteins; cell death; and eventual calcification. Environmental influences from infections, diabetes, and stress can accelerate atheroma formation, whereas statins may facilitate atheroma regression (Chapter 2). The insulin/IGF-1 system that modulates life span in flies and worms is also at work in many aspects of atherogenesis (Fig. 1.3B). Animal models vary in susceptibility to arterial lesions. Macaques, chimpanzees (Finch and Stanford, 2004; Wagner and Clarkson, 2005), and rabbits (Yanni, 2004) are more vulnerable to atheroma induction by diet and stress than lab rodents (Moghadasian, 2002). The apoE-knockout mouse has extreme susceptibility to atheromas, in asso- ciation with its extreme hypercholesterolemia (Rauscher et al, 2003). The myocardium is altered during aging through inflammatory processes that can interact with arterial changes. Left ventricular stiffness increases progressively with aging (decreased ‘compliance’) and slows the diastolic of filling rate by up to 50% by age 80 (Brooks and Conrad, 2000; Lakatta and Levy, 2003a,b; Meyer et al., 2006). The stiffness is due to ventricular wall thickening and interstitial myocardial fibrosis, and possibly collagen cross-linking through nonenzymatic glycation. Fibrosis is very common during mammalian aging and deeply linked, if not intrinsic, to general inflammatory processes in aging (Thomas et al, 1992). TGF-β1 signaling pathways that regulate collagen synthesis are implicated in myocardial fibrosis. TGF-β1 deficiency (+/− heterozygote knockout) attenuated the age-related increase of left ventricular fibrosis, improved cardiac performance, and possibly increased life span (Brooks and Conrad, 2000). Myocardial stiffness is attenuated in
  • Inflammation and Oxidation in Aging and Chronic Diseases 19 humans during diet restriction in at least one study (Section 3.4.1). Conversely, transgenic mice with increased systemic TGF-β1 developed premature left ventricular fibrosis with increased levels of TIMPS (tissue inhibitor of metalopro- teinase, also implicated in arterial aging) (Seeland et al, 2002). Mitochondrial DNA changes in the myocardium also merit mention because of their interactions with ischemia and oxidative stress. Additionally, myocardial mitochondrial DNA deletions (mtDNA4977 , nt 8469–13,447) increase modestly after age 60 (up to 7 per 10,000 mitochondria). Ischemic hearts can have >200- fold more mtDNA deletions (Botto et al, 2005; Corral-Debrinski et al, 1992), which is attributed to the oxidative stress from ischemia. Because the DNA deletion impairs mitochondrial function and increases respiratory chain stress, a vicious cycle is hypothesized to cause further mitochondrial damage. Single base changes (point mutations) also increase with aging in a mutational hotspot (nt 16,025–16,055, control region) in cardiomyocytes, but not buccal epithelial cells, with indications of clonal expansion (Nekhaeva et al, 2002). Besides these aspects of myocardial aging, there are many other aging changes, as well as compensatory mechanisms that go beyond this discussion. At the behavioral level, and of great importance to human aging, are complex social and psychological links to vascular disease and hypertension (‘social etiology’) (Berkman, 2005; Marmot, 2006; Sapolsky, 2005). Social stress also accelerates vas- cular changes in primates and rodents (Andrews et al, 2003; Henry et al, 1993). Complex social interaction during aging has not been defined in animal models. Immunity declines in complex ways during aging: instructive immunity weak- ens concurrently with increased inflammation in most tissues and chronic diseases. Both changes may contribute to the decreased resistance to oppor- tunistic infections, incidence and severity (Akbar et al, 2004; Miller, 2005; Pawelec et al, 2005; Weksler and Goodhardt, 2002). As examples, the elderly suffer 90% of the influenza deaths, while HIV has a shorter latency in the eld- erly, reviewed in (Olsson et al, 2000). The decreased resistance is associated with various dysfunctions of systemic and tissue immune mechanisms: the atten- uation of adaptive (instructive) immunity and the hyperactivity of acute phase host defense processes. Aging of the adaptive immune responses may be very gradual in populations of humans and lab animals with low burdens of infec- tion and inflammation, and good nutrition. Nonetheless, naive T cells progres- sively decrease at the apparent expense of memory T cells (CD4 and CD8) (Haynes, 2005; Linton and Dorshkind, 2004; Miller, 2005; Pawelec et al, 2002). At birth, nearly all T cells express CD28, a major T cell-specific co-stimulator that binds to sites on antigen-presenting cells and activates IL-2 transcription, cell adhesion, and other critical T-cell functions. CD28 is progressively lost during aging (Merino et al, 1998; Pawelec et al, 2005; Trzonkowski et al, 2003). The loss of CD28+ T cells is attributed to chronic antigenic stimulation over the life span. Accelerated loss of CD28 T cells is observed in young HIV patients and is modeled in cultured T cells (Posnett et al, 1999). The CD8+ CD28− T cells are resistant to apoptosis and are considered ‘fully differentiated.’ During influenza inflections,
  • 20 The Biology of Human Longevity the elderly have decreased cytotoxic T-cell activity in association with shifted cytokine profiles (T-helper type 2 dominance) (McElhaney, 2005). Declines of thymus function begin before maturation (Krumbahr, 1939; Min et al, 2005; Steinmann, 1986). Infections and malnutrition in the early years can impair thymus development with later consequences to immunity (Moore et al, 2006; Savion, 2006). Striking examples come from West Africa. In rural Gambia, seasonal infections during childhood alter T-cell functions with correspondingly increased adult mortality (Moore et al, 2006). In Guinea-Bissau, a low small thymus is associated with increased mortality from infections (Aaby et al, 2002). These and other environmental effects on immunity during development are discussed in Section 4.6.2. Between puberty and mid-life, adipocytes gradually replace the lymphocytic perivascular space. These gross changes are preceded by regression of the thymic epithelium and can be delayed by castration before puberty (Chiodi, 1940; Min et al, 2006). After maturation, the thymus continues to generate T cells throughout life, although at lower levels (Douek et al, 2000; Hakim et al, 2005). Immune homeostatic mechanisms decline during aging; e.g., T-cell recovery after chemotherapy is greatly reduced by age 50 (Hakim et al, 2005; Hakim et al, 2005). Extra-thymic aging changes include lower bone marrow production of lymphopoietic progenitor cells, possibly due to decreased growth hormone and IGF-1 (Hirokawa et al, 1986; Linton and Dorshkind, 2004). Most immunologists agree that thymic involution is multi-factorial and that immune aging is not reversed by simply restoring GH, IGF-1, or other hormones that change with aging (Chen et al, 2003; Min et al, 2006). The adverse effects of infections and malnutrition on thymic development may extend to other aspects of immunity. The major shifts from virgin T cells to memory T cells during the life span are attributed to exposure to common infections, environmental antigens, and auto- antigens. Cytomegalovirus (CMV), an endemic ß-herpes virus that is a common infection in childhood, may be a general factor in the clonal depletion of CD28+ T cells (Koch et al, 2006; Pawalec et al, 2005). Up to 25% of the CD8 T cells in older healthy humans are CMV-specific (Khan et al, 2002) and are approaching replicative senescence (Fletcher et al, 2005). A proposed ‘immune risk phenotype’ of aging is characterized by (1) CMV-seropositivity; (2) inverted ratios of CD4:CD8 <1 (unlike the normal CD4 excess in healthy young adults); and (3) increases in ‘fully differentiated’ CD8+ CD28− effector T cells, which have shortened telomeres and limited proliferation (Olsson et al, 2000; Pawelec et al, 2005). Elderly with ratios of CD4:CD8 <1 have 50% higher mortality in two populations: the Healthy Ageing Study (Cambridge UK) (Huppert et al, 2003) and the OCTO and NONA Longitudinal Studies ( Jönköping, Sweden) (Wikby et al, 2005). These T-cell shifts decrease resistance to new infections. The greater vulnerability of elderly to influenza may be attributed to imbalances of central memory T cells over the effector memory T cells that mediate virus-specific IFN production (Kang et al, 2004). CMV-seropositive elderly who responded poorly to influenza vaccine also had more CD28- lymphocytes (Effros, 2004; Trzonkowski et al, 2003) and 2-fold
  • higher IL-6 and TNFα (Trzonkowski et al, 2003). The higher cytokine production during aging in immune responses may extend to other classes of T-cells (O’Mahony et al, 1998) and may be a factor in the strong age trend for elevated cytokines (Section 1.8.1). Besides CMV, many other infections influence the ‘immune aging pheno- types.’ Chronic immune activation can accelerate ‘aging’ of T-cell functions, as observed in infections by HIV (van Baarle et al, 2005) and nematode parasites (Borkow et al, 2000) (Section 2.7.1). As noted previously, childhood infections affect the thymus and impair immunity and increase mortality (Section 4.6). As another example, mice with genetically determined elevations of memory T-cells have shorter life spans and higher prevalence of tumors at middle age (Miller, 2005). Chronic immune activation also increases ‘bystander’ damage (Section 1.4.3) (Query II). We may anticipate that outcome of immune aging depends on gene-environment interactions with inflammatory gene variants, particularly the proinflammatory IL-6 and the antiinflammatory IL-10 (Caruso et al, 2004) (Section 1.3.2). The strong role of the antigenic environment on immune aging is included in the framework of Fig. 1.2A and extends to direct involvement of T-cells with unstable atheromas (Section 2.2.2). Telomere erosion is implicated in immune aging in association with the reduced proliferation of T cells (Effros, 2004). In peripheral lymphocytes, telomeres shorten by 50 base pairs per year across the life span against initial telomere lengths at birth of about 15,000 base pairs (Hathcock et al, 2005). Telomeres are shorter in primed T-cell subsets, especially the ‘effector memory’ T cells (Akbar et al, 2004). Telomere loss may eventually activate gene regulatory programs leading to cell death (apoptosis) or a post-mitotic state (clonal senescence; considered equivalent to the Hayflick limit; see below). Telomerase reactivation is thought to be adaptive for clonal expansion without rapid clonal senescence. However, T cell proliferation does not always cause telomere erosion, because immune stimulation of B and T cell proliferation can induce telomerases (Akbar et al, 2004; Hathcock et al, 2005; van Baarle et al, 2005). Other evidence argues against telomere erosion as a general mechanism in immune aging (Miller et al, 2000); e.g., although mice have much longer telomeres than humans, mouse T cell proliferative aging is faster. Much is unknown about enzymes that mediate telomere replication, which differs between immune cell types and animal species. In contrast to the decline of antigen-driven immunity, inflammatory processes in many tissues progressively increase during aging, e.g., muscle, fat, brain (Section 1.8.1). Inflammatory gene expression increases in these and other tissues. Blood IL-6 and C-reactive protein generally increase during aging in human pop- ulations, although much of the increase is associated with vascular disease. Tissue-specific macrophages are prominent in atheromas (‘foam cells’), Alzheimer disease (microglia), and bone (osteoclasts). Apart from these degenerative dis- eases, studies of circulating macrophages from aging humans and rodents are puzzlingly inconsistent about the direction and type of aging changes (Finch and Inflammation and Oxidation in Aging and Chronic Diseases 21
  • Longo, 2001; Pawelec et al, 2002; Wu and Meydani, 2004). Despite blood IL-6 ele- vations, induction of IL-6 in response to LPS (gram-negative bacterial endotoxin) decreases with age in peritoneal macrophages (Stout and Suttles, 2005), but increases with age in brain microglia (Xie et al, 2003; Ye and Johnson, 1999). Lastly, we should be mindful that decreased system-level and integrative functions (‘organ reserves’) contribute to mortality independently of specific immune subsystems. The declining ‘vital capacity’ of lungs (Janssens and Krause, 2004; Meyer, 2005) is strongly associated with survival in general and resistance to respiratory infections. In the Framingham Study, mortality risk at age 50–59 varied inversely with the lung vital capacity (Ashley et al, 1975; Finch, 1990, p. 563). Smoking, which decreases pulmonary volume and respiratory functions, increases vulnerability to influenza and pneumonia (Murin and Bilello, 2005). In the Cardiovascular Health Study of persons 65 years and older, smokers had a 50% higher risk of hospitalization for pneumonia and a 28% higher mortality in the 2.4 years after discharge (O’Meara et al, 2005). Moreover, CMV and other chronic infections may deplete the bone-marrow-derived endothelial progenitor cells that mediate vascular repair (Section 2.7.3). Thus, the decreased resistance to infectious disease should be analyzed in terms of systemic physiology and the ecological life history of exposure in infections and inflammogens (Chapters 2–4), which are subject to gene-environment interac- tions throughout the life history (Chapters 4 and 5). Female reproductive senescence is due to the exhaustion of ovarian oocytes in all mammals examined (Finch, 1990, pp. 165–167; Gosden, 1984; vom Saal et al., 1994; Wise et al, 1999). Oocyte numbers are fixed during development by the cessation of primordial germ cell proliferation. Oocyte loss begins before birth and continues exponentially, like radioactive decay (Faddy et al, 1992). Recent evidence refutes the possibility of continuing de novo oogenesis from cir- culating stem cells (Eggan et al, 2006). Less than half of the original stock remains by puberty. The rate of oocyte loss is slowed by diet restriction, which alters hypothalamic controls of the gonadotrophins (Chapter 3, Fig. 3.17). Fecundity declines long before the failure of ovulation due to oocyte depletion, with marked reduction by age 35 years in women; lab rodents aged 8–12 months are culled as ‘retired breeders’ by production colonies. With the loss of ovarian follicles, the production of estrogens and progestins decreases sharply in human menopause, causing hot flushes, as also observed in macaques (Appt, 2004; Nichols and O’Rourke, 2005). Ovarian steroid loss is implicated in the post-menopausal increase of vascular disease and may interact with vascular inflammatory processes. In males, androgen levels show trends for decline, but more sporadically than in females. Estrogen replacement (hormone therapy), while controversial, appears to be health protective for some women (Section 2.9.4). Androgen replacements may benefit arterial disease, cognition, and glycemic control (Harman, 2005; Jones et al, 2005; Liu et al, 2004; Morley et al, 2005). The sharp rise of vascular disease during middle age is an example for the declining strength of natural selection during aging (evolutionary perspec- tives, below). 22 The Biology of Human Longevity
  • Bones and joints degenerate broadly during aging in mammals in processes that involve inflammatory regulators (Section 1.8). Osteoporosis (bone mineral resorption) occurs through an imbalance of production by osteoblasts versus resorption by osteoclasts. The inflammatory system is involved in bone resorption. First, osteoclasts are of macrophage/monocyte lineage. Then, bone resorption is stimulated by inflammatory cytokines (IL-1, TNFα) (Clowes et al, 2005; Tanaka et al, 2005). Osteoporotic bone loss accelerates after menopause and can be atten- uated by estrogen replacement. In some contexts, estrogen has anti-inflammatory activities (Amantea et al, 2005; Thomas et al, 2003) (Section 2.10.4). Osteoarthritis is a focal, age-related inflammatory lesion in the joints that can be painful (Section 1.7). Mechanical pressures activate inflammatory cells and catabolic responses of the articular chondrocytes that cause matrix loss and accumulation of AGEs. Brain-aging atrophic changes are manifest soon after maturation, in healthy humans by age 30 y and rodents aged 10 m (Finch et al, 1993; Teter and Finch, 2004). The volume of the brain as a whole shrinks by about 0.5%/year in normal humans across the adult age range, 20–98, and is accelerated by Alzheimer dis- ease to about 1%/y (longitudinal MRI) (Burns et al, 2005; Fotenos et al, 2005). The volume of the hippocampus, which is critical to declarative memory, also shrank linearly in healthy elderly observed over 6 y (Cohen et al, 2006). Neuron loss during aging is more limited than once widely presumed (‘neu- romythology’) (Finch, 1976; Gallagher et al, 1996; Rasmussen et al, 1996; Teter et al, 2004; Tomasch, 1971). During normal human aging, the total number of cortical neurons does not change, but neuronal size shrinks. Small cortical neu- rons increase, while the numbers of large neurons decreases reciprocally (Terry et al, 1987) (Fig. 1.7A). Synaptic loss parallels brain atrophy and neuron shrinkage, with progressive decreases in the presynaptic protein synaptophysin in the normal aging cerebral cortex (Fig. 1.7B) and dopamine D2 receptors in the cortex and striatum (Fig. 1.7C, D) (Morgan et al, 1987; Reeves et al, 2002; Suhara et al, 2002; Wong et al, 1997). Other receptors, however, may increase—e.g., dopamine D1 receptor (Morgan et al, 1987)—possibly as a compensatory response. The extent of synaptic loss approximates 1% loss per year after age 20. Rodent brains show similar changes in dopamine receptors, scaled to their shorter life span. By the mean life span, synap- tic atrophy reaches 30–50%, independent of Alzheimer, stroke, or other clinical con- ditions in lab rodents and humans. Nonetheless, neuron cell death is minimal in cortex and possibly other brain regions to advanced ages, absent Alzheimer changes. Even at later ages, neuron loss in aging memory-impaired rats is modest or sporadic in brain regions afflicted by Alzheimer disease (Rasmussen et al, 1996; Rapp and Gallagher, 1996). However, sporadic neuron loss may arise from various stressors (Landfield et al, 1977; Meaney et al, 1988) or toxins (Section 3.2.3; Finch, 2004b). In rodents, D2 receptor loss in aging is attenuated by diet restriction (Chapter 3, Fig 3.17) while neuronal atrophy is reversed by nerve growth factors (Smith et al, 1999). The generality of synaptic atrophy during middle age in the absence of neuron loss distinguishes these changes from the subgroups at later ages that develop aggressive neurodegeneration during Alzheimer disease. Inflammation and Oxidation in Aging and Chronic Diseases 23
  • 24 The Biology of Human Longevity 20 30 40 50 60 70 80 90 Y 1200 1000 800 600 400 200 Smallneurons 30 40 50 60 70 80 9020 Y 20 30 40 50 60 70 80 90 Y A1700 1500 1300 1100 900 700 500 700 500 600 400 300 200 100 800 Human cerebral cortex Allneurons Largeneurons 70 80 90 60 100 0 B 20 40 60 80 Y Presynapticterminalsper100sqµm r=−0.708 p=0.0001 Human cerebral cortex FIGURE 1.7 For legend see page 25. 100 200 300 20 40 60 80 D-2Receptordensity:Humanstriatum Y 400 100 120 160 28 M2083 140 80 60 D-2Receptordensity:Mousestriatum 40 C Mouse striatum Human striatum
  • 40 0 10 20 30 40200 D 60 80 Y D-2receptordensity:human(PET) Human striatum Inflammation and Oxidation in Aging and Chronic Diseases 25 FIGURE 1.7 (continued) Normal brain aging: neuronal atrophy, glial hypertrophy, declining blood flow in brains without Alzheimer disease or ischaemic damage. A. Total cerebral cortical neuron numbers do not change. However, the numbers of small neurons increase inversely with decreased numbers of large neu- rons, apparently due to atrophy of large neurons (Masliah et al, 1993; Terry et al, 1987). B. Presynaptic terminal density (synaptophysin immunostaining) shows progressive decline of about 0.75%/y (Terry et al, 1987), possibly associated with neuronal atrophy. C. Dopamine (D2) receptor loss in human and mouse (ligand binding, postmortem), approximating 1%/y after age 20. (Redrawn from Morgan et al, 1987a.) D. In vivo D2 receptor sites (positron emission tomography/PET) in caudate of normal humans declined 1%/y after age 20 (Wong et al, 1997). E. Aging trends of synaptic density (panel B above), cerebral blood flow (Fig. 1.20) (Amano et al, 1982), and astrocyte volume in brains without Alzheimer disease or ischaemic damage (Hansen et al, 1987). Cerebral cortex from the same source and evaluated by the same criteria for health was used for studies of astrocyte volume and synaptic loss (Panel B). 100 0 25 50 75 4020 60 800 E Y %,young Astrocyte vol. Blood flow Synapse density Human cerebral cortex
  • 26 The Biology of Human Longevity Age-related synaptic atrophy (15–30%), while modest relative to Alzheimer disease, plausibly contributes to declines in complex brain functions. Memory capacity declines progressively in humans and rodents, with no evident pathol- ogy (Albert, 2002; Rajah and D’Esposito, 2005; Rosenzweig and Barnes, 2003; Woodruff-Pak, 2001). In the hippocampus, a seat of declarative memory, synapse loss is extensive. The hippocampus receives input from the cerebral cortex from the perforant pathway, which is much more damaged during Alzheimer disease than normal aging (Nicholson et al, 2004; Rosenzweig and Barnes, 2003). Some functional deficits may be linked to synaptic atrophy. The D2 receptor loss during aging (Fig. 1.7D) correlated with performance on tasks dependent on the frontal cortex, e.g., the Stroop Color-Word Test interference score (Volkow et al, 2000). Other deficits may be due to the deterioration of myelinated pathways (white matter), which mediate high-speed exchanges between brain regions. Microglial activation may be a factor in white matter changes during middle age as seen by brain imaging (Bartzokis, 2004; Bartzokis et al., 2003, 2004, 2006; Burns et al., 2005) (Fig. 1.8B). ApoE4 carriers have accelerated myelin deterioration (Bartzokis et al., 2006), consistent with the ‘proinflammatory’ associations of the E4 allele (Section 1.3). White matter inflammatory changes may contribute to the usual slowing of information processing and the decreased multi-tasking by middle age (Bashore, 1994; Madden, 2001; Verhaeghen and Cerella, 2002). Multi-tasking becomes progressively impaired during aging. A striking example is the impairments by middle age in memorizing words while walking an irreg- ular course (Li et al, 2001). Multi-tasking depends on high-speed processing across multiple circuits, which slows progressively during normal aging (Bashore and Ridderinkoff, 2002; Maeshima et al, 2003; Ylikoski et al, 1993). These processes suggest why few professional athletes remain competitive over the age of 40 and why driving errors increase with aging (Campagne et al, 2004). Skeletal muscle atrophy during aging (‘sarcopenia’), a major factor in frailty (Dow et al, 2005), may be due to motor neuron aging. The atrophy of aging muscle resembles denervation atrophy and is partly reversed by electrical stimu- lation (Dow et al, 2005). The major role of motor neuron age in skeletal muscle aging was shown by a powerful transplantation experiment: when reinnervated in young rat hosts, the 32-month-old muscle grafts regained full contractile strength (Carlson et al, 2001). Aging muscle also accumulates mitochondrial DNA mutations in association with muscle fibers deficient in cytochrome oxidase (COX) (Brierley et al, 1998; He et al, 2002; Kopsidas et al, 2002). However, COX- deficient fibers are relatively rare (0.1–5%), and the deficiency does not extend through the entire fiber (Brierley et al, 1998; Frahm et al, 2005). Thus, muscle mtDNA mutations may contribute less to muscle aging than motor neuron impairments. Impaired axoplasmic flow by motor neurons in the sciatic nerve (Goemaere-Vanneste et al, 1988) and spinal projections (Frolkis et al, 1985; McQuarrie et al, 1989) could be major causes of the reduced neurotrophic support. Axoplasmic flow also decreases during aging in the central projections
  • (De Lacalle et al, 1996; Geinisman et al, 1977). Cell-level gene expression may identify global or cell-specific impairments in biosynthesis that could cause diverse manifestation of synaptic atrophy. What may cause the atrophy of neurons during aging? My lab is studying the increase of glial inflammatory changes that are concurrent with neuronal atrophy, in healthy humans, rodents, and monkeys (Morgan et al., 1999; Finch et al., 2002). Astrocytes and brain macrophages (microglia, of bone marrow lineage) are activated by middle age (Fig. 1.8A). The increased volume of astrocytes mainly represents cell hypertrophy. The total number of astrocytes does not increase during normal aging (Bjorklund et al, 1985; Finch et al., 2002; Long et al, 1998), although there are more and larger fibrous astrocytes with thick, GFAP containing processes (Hansen et al., 1987). GFAP is a cytoskeletal protein (intermediate filament) that increases with aging (Nichols et al., 1993) in parallel with increased astrocyte volume (Fig. 1.7E). The age-related increase of GFAP expression can be considered as an inflammatory response (Morgan et al, 1999). GFAP transcription increases in response to oxidative stress and inflammatory stimuli, possibly through redox sensitive elements (NF-1/NF-κB) in the upstream promoter (Morgan et al, 1997b, 1999). Diet restriction attenuates the increase of GFAP transcription and protein levels with aging (Morgan et al, 1999) in parallel with attenuating synaptic atrophy during aging (Chapter 3). Many other brain changes during aging are associated with inflammatory processes (Section 1.8.1). We hypothesize that glial activation during aging is an inflammatory response to oxidative damage that, in turn, causes synaptic atrophy (Rozovsky et al., 2005). Microglia activation during aging is attenuated by diet restriction (Chapter 3, Fig. 3.19). We are testing these concepts by growing cultures of astrocytes from aging rats, which show age deficiencies in support of neuronal outgrowth (Fig. 1.9). The age deficiencies are associated with increased expression of GFAP and are inversely associated with secretion of laminin and other extracel- lular substrates. Conversely, we can induce an age-like phenotype in young astrocytes by increasing the cell levels of GFAP, with correspondingly less sup- port of neurite ourtgrowth. These results show the close links of GFAP expres- sion to astrocytic support of neurite outgrowth over a 2-fold range (Rozovsky et al, 2005). Alzheimer disease (AD) differs from these normal brain aging changes (Section 1.6) by severe neurodegeneration in memory circuits with remarkable selectivity. The hippocampal pyramidal neurons of the CA1 field are devastated, while nearby granule neurons are relatively unscathed. Neuronal vulnerability to endogenous or exogenous insults must ultimately depend on gene expres- sion patterns acquired during cell differentiation. AD is rare before age 60 and increases exponentially thereafter with a doubling rate of about 5 years (Fig. 1.10) (Kawas and Katzman, 1999; Mayeux, 2003). By age 80, the AD prevalence approaches 50% in some populations but may be less than 15% in others. These huge differences are unexplained. Inflammation and Oxidation in Aging and Chronic Diseases 27
  • FIGURE 1.8 White matter (myelinated pathway) aging. A. Microglial activation in cortico-striatal myelinated tracts of aging F344 rats. Note the increased immunostaining (dark area) in 24 m markers of microglial activation; MHC-2, antigen of activated macrophage/microglia; CR3, complement receptor. (From Morgan et al., 1999.) B. Myelin integrity declines with aging in the corpus callosum (subcortical white matter) of normal brain (o) after age 50, with greater deterioration in Alzheimer disease ( ). Determined from the transverse MRI relaxation rate signal (R2), a measure of myelin integrity (Bartzokis et al., 2004). 12 18 17 16 15 14 13 10 807060504030200 B Y Myelin organization (MRI) Normal AD 24 M3 M OX 6 OX 42OX 6 A OX 42
  • Manipulation of GFAP and neurite outgrowth (unlesioned co-culture) Age impairment in neurite outgrowth is reversed by RNAi BA C Neurite outgrowth is inhibited in co-cultures with young astrocytes transfected with GFAP cDNA MAP-5immunoreactiveneurites (%ofyoungmutRNAi) MAP-5immunoreactiveneurites (%ofcontrol) 100 50 0 mutRNAi RNAi young mutRNAi RNAi old 120 100 80 60 40 20 0 0.10 0.3 GFAP cDNA transfected (µg/well) Inverse relationship between GFAP expression and neurite outgrowth 60 40 20 0 −20 −40 −60 −80 −40 0 40 80 120 GFAP (%) MAP-5(%) Age effect O RNAi Y cDNA Y RNAi * * FIGURE 1.9 Astrocyte aging influences the growth of embryonic neurons. In the heterochronic glial- neuronal model, astrocytes are cultured from young adult and old rat cerebral cortex and then over- lain with embryonic (E18) cortical neurons (Rozovsky et al, 2005). Neuronal outgrowth on old astrocytes is markedly slower relative to growth on young astrocytes. The age effect on neurite out- growth is reversed by downregulating GFAP, an astrocyte cytoskeletal protein that increases with aging (Nichols et al, 1993) in parallel with increased astrocyte volume (Fig. 1.7E). The age-related increase of GFAP expression can be considered an inflammatory response during aging, and is attenuated by diet restriction (Morgan et al, 1999) (Fig. 3.17B, Section 3.5.2). The lower panel summarizes exper- iments that manipulated GFAP bidirectionally in young and old astrocytes, with corresponding inverse effects on neuriote outgrowth (MAP-5 marker).
  • FIGURE 1.10 Alzheimer disease. A. Alzheimer prevalence increases after age 65 with a doubling time of about 5 y, which is faster than the Gompertz mortality rate doubling time of 8 y. Populations vary widely in dementia prevalence by 80 y (Kawas and Katzman, 1999). B. Senile (neuritic) plaque of Alzheimer brain showing abnor- mal neurites and activated glia. Courtesy of Christian Pike (USC). 0.1 0.01 1 10 100 Prevalence(%) 65 A Y9085807570 CANADA E. BOSTON EURODEM FRAMINGHAM HISAYAMA JORM KAOHSIUNG SEATTLE-JAPANESE SHANGHAI TOKYO
  • Inflammation and Oxidation in Aging and Chronic Diseases 31 A characteristic of AD is extensive deposits of extracellular senile plaques containing fibrillar Aβ1-42 (Fig. 1.10B) and intraneuronal aggregates of neu- rofibrillary tangles containing hyperphosphorylated tau. A major hypothesis is that AD is driven by excess production of the 42 amino acid long amyloid β- peptide (Aβ1-42 ) (Section 1.6). The diagnosis of Alzheimer disease is made by a threshold density of plaques, tangles, and neuron loss in the cerebral cortex and hippocampus. The diagnosis may be adjusted for age because of the increase of neurofibrillary tangles during aging. Non-demented elderly show accelerating increases of tangles to levels that overlap with early clinical AD (Braak and Braak, 1991; Price and Morris, 1999) and also show modest corti- cal atrophy with aging (Anderton, 1997; Launer et al, 1995). At very advanced ages, plaque and tangle accumulations may overlap with criteria for AD. Amyloid accumulation in animal models is slowed by anti-inflammatory drugs (Chapter 2) and diet restriction (Chapter 3). Rodents are valuable exper- imental models to study human AD transgenes because they do not accumu- late brain amyloid deposits during aging. Amino acid substitutions in the rodent Aβ sequence (Johnstone et al, 1991) render it less able to aggregate into fibrils and possibly less toxic (Boyd-Kimball et al, 2004). In other vertebrates, the Aβ sequence is remarkably conserved—fish to primates to humans. Aging dogs and macaques accumulate Aβ deposits that resemble senile plaques of AD. However, aging chimpanzees, our closest ancestor, have negligible AD-like changes during aging (Section 6.3) (Finch and Stanford, 2004). The chimpanzee does not have a common risk factor in AD, the apoE4 allele, which evolved in humans. 1.2.3. Cultured Cell Models and Replicative Senescence Unlike cancer cells, diploid somatic cells typically have a finite capacity for prop- agation during serial culture, shown first for skin fibroblasts (Hayflick and Moorhead, 1961). After a finite number of subcultures (population doublings), cell division ceases and cultures are considered senescent. The ‘Hayflick’ phe- nomenon extends to many cell types, including vascular endothelia and lym- phocytes (Campisi, 2005; Cristofalo et al, 2004; Hayflick, 2000). Although the end-phase cultures are considered ‘senescent,’ post-replicative cells survive many months if media are refreshed (Matsumura et al, 1979). In fact, senescent cultures are highly resistant to apoptosis. Resistance to apoptosis may link back to the insulin pathways that modulate life span (Fig. 1.3), because senescent cell cul- tures have decreased endocytic uptake of the IGF-binding protein IGFBP-3 (Hampel et al, 2005). It is cogent to Query I that senescent cultures of fibroblasts and other cell types show increased inflammatory factors including COX-2, IL-1, MMP-3, collagenase, TIMP-1 (tissue inhibitor of metaloproteases) (Han et al, 2004; Parrinello et al, 2005; West et al, 1989; Zeng et al, 1996). These same changes arise in atheromas and, moreover, are blocked in senescing cultures by
  • 32 The Biology of Human Longevity the COX-2 inhibitor NS398 (Han et al, 2004) (Chapter 2). Inflammatory factors secreted by replicatively senescent cells are implicated in focal tissue remodeling in the progression of pre-malignant cells (Campisi, 2005). Individual cell variations in replicative potential are associated with variable telomere length (Martin-Ruiz et al, 2004). It is not known if telomere hetero- geneity causes the daughter cell differences in proliferative potential, which range from 0 (growth arrest) to 15 or more replications (Matsumura et al, 1985). Somatic cell replicative heterogeneity may contribute to the remarkable differ- ences of individual life span in twins and in highly inbred worms (Finch and Kirkwood, 2000) (Section 5.2). Contrary to earlier conclusions, adult age up to 92 years does not change the Hayflick limit of skin fibroblasts (Cristofalo et al, 2004; Goldstein et al, 1978; Smith et al, 2002). However, cells from embryos or children have 2-fold higher Hayflick limits (Martin, 1970). Thus, the major effect of age on the cell senes- cence model is before maturation. In vivo exposure to oxidative stress may be a factor in the reduced proliferation of cells from diabetics (Goldstein et al, 1979). Moreover, the standard protocol of culturing cells for aging studies in ambient air (20% oxygen) is grossly unphysiological. When mouse cells were ‘aged’ at 3% oxygen, closer to the tissue levels, their replicative potential was greatly increased (Parrinello et al, 2003). Nonethless, even under the standard culture conditions, in species comparisons, resistance of cultured fibroblasts to oxidative stress correlates with life span (Kapahi et al., 1999). We may anticipate fruitful further analysis of species differences in resistance to oxidative stress that might, in turn, inform about in vivo vulnerability to bystander effects from inflammatory processes. 1.2.4. Invertebrate Models The fly and worm models are enabling highly successful studies of genetic influences because their gene regulatory systems of early development are known in detail (Davidson, 2006; Giudice, 2001; Grant and Wilkinson, 2003). Mutations modify longevity in association with altered mortality rate accelera- tions (Chapter 5). Some mutations that modify aging involve insulin-like sig- naling pathways and fat depots (Fig.1.3A). These convergences suggest the importance of energy regulation to aging, as well as to development. The energy-regulating gene circuits have persisted during descent from shared ancestors more than 650 million years ago. Although the causes of death in fly and worm are not well defined, the causes do not include tumors or other abnormal growth during aging. The lab worm C. elegans naturally grows among the roots of plants. Propagation by self-fertilization eliminates more genetic variation than is possi- ble with inbred laboratory mice (Johnson et al, 2005). Free-living larva hatch about 24 h after fertilization, followed by rapid development through larval stages (L1-L4) and maturation by 72 h. If food is limited, or population density
  • Inflammation and Oxidation in Aging and Chronic Diseases 33 is high, the larval development may be arrested for up to 2 m in the dauer larval stages. Dauer larvae cease feeding and utilize fat depots; body movements decrease, but stress resistance increases (Kimura et al, 1997). With improved conditions, dauer larvae complete maturation and proceed to normal life spans. Worm life history has four stages lasting 2–3 w (Huang et al, 2004): I, active egg production by self-fertilization in the first 4 d (Bolanowski et al, 1983; Herndon et al, 2002), followed by several post-reproductive stages: II, post- reproductive, with vigorous movements; III, dwindling movement leading to the cessation of feeding; and IV, morbidity with little movement and accelerating mortality. Most eggs are produced during the first 4 days (Croll et al, 1977; Johnson, 1987; Klass, 1977). While life spans in different genotypes and environments are well docu- mented, less is known about the cellular changes and the pathology of aging. Cell death is not obvious during aging, despite the lack of somatic cell replace- ment. C. elegans is famous for its almost invariant cell number. Neurons look normal in ultrastructure studies throughout life, including neurons of slowed and decrepit worms (Herndon et al, 2002). However, muscle cells deteriorate in the body wall and in the pharynx, which grinds up bacteria that are the diet (Herndon et al., 2002). Lipids, lipofuscins (aging pigments), and lysosomal hydrolases accumulate in muscle and intestine cells (Bolanowski et al, 1983; Epstein et al, 1972; Garigan et al, 2002; Herndon et al, 2002), implying defects in catabolic pathways. Old worms are less resistant to pathogenic bacteria and show shorter latent period after infection (Kurz et al, 2003; Laws et al, 2004). Moreover, aging worms become constipated from bacterial packing in the intestine, which may induce oxidative damage. The usual diet of the bacteria Escherichia coli strain OP50 is considered by some to be mildly toxic; life spans are longer on heat-killed bacteria or other media (Section 2.3.2, Section 5.5.2). Long-lived mutants in insulin-like signaling (age-1) have delayed con- stipation (Section 5.5.2). Although these worms are isogenic, constitutive variations in the levels of gene expression arise during development that influence later outcomes of aging (Finch and Kirkwood, 2000). Individual worms vary in the duration of these stages and in rates of aging. This extensive variability may be considered to extend variations present at younger ages in egg laying, feeding, and spontaneous movements (Finch, 1990, p. 560; Finch and Kirkwood, 2000). Individual declines of pharyngeal pumping and body movement were strongly correlated with life span in wild-type and longevity mutants (Chapter 5). For example, when fast pumping is maintained one day longer, the odds ratio for death by or later than a specified date is 1.7-fold greater (Huang et al, 2004a). The levels of expression of a stress-protective gene (hsp-16.2) in young worms predicted future life span, over a 2-fold range (Rea et al, 2005). This first example of individual difference in gene expression in the worm model supports the role of epigenetic variations arising during development that may ultimately represent chance variations in the assembly of the multiple proteins present in transcription complexes (Finch and
  • 34 The Biology of Human Longevity Kirkwood, 2000). In another model, cultured mammalian cells with a reporter gene did not respond synchronously or to the same level to a diffusible inducer (Zlokarnik et al, 1998). The fly is a more complex animal with a beating heart. At 25 ˚C, early devel- opment takes 24 h to larval hatching. Three mobile feeding larval stages (LI- LIII) take 7 more d to pupation. During the 4-day pupal stage, without feeding or movement, the adult body is formed from the imaginal disks (metamorphosis). Adult life spans are about 40 d. Adult flies can over-winter, with extended life span from cool temperature and shorter photoperiods (Flatt et al, 2005; Finch, 1990, p.313; Schmidt et al, 2005). Unlike nematodes, the fly does not have alternate larval stages equivalent to the non-feeding dauer. Juvenile hormone (JH), a series of steroid-like molecules, influences or regulates growth of all developmental stages, particularly the timing of molts and metamorphosis, and also adult dia- pause (Flatt et al, 2005). JH synthesis is regulated by insulin-like peptides secreted by neurons (Section 5.4). JH also regulates stress resistance and immune responses that are like innate immunity of vertebrates. Female egg-laying declines exponentially after a fairly stable phase, also observed in the medfly (Ceratitis capitata) (Novoseltsev et al, 2004). Both species have post-reproductive phases that only weakly correlated among indi- viduals with the cessation of egg-laying. As with the worm, somatic cells are not replaced. Major damage is accumulated to the brittle exoskeleton from wear- and-tear (Finch et al, 1990). Unlike the worm, the aging fly shows some indi- cation of neuron loss, in the mushroom body (Technau, 1984). The fat body, a key organ of energy reserves and immune function, gradually atrophies (Finch, 1990, p. 63). Apoptosis with DNA fragmentation increases in flight muscles and fat body (Zheng et al, 2005). The heart rate slows during aging and arrests more easily under the stress of electrical pacing, aging sharply (Wessells et al., 2004) (Section 5.6.3, Fig. 5.7). Insulin-signaling mutants with increased life span have delayed cardiac aging (Chapter 5). Little is known about vasculature of aging flies; other aging insects show indications of circulatory blockage (Arnold, 1961, 1964; Finch, 1990, p. 65). 1.2.5. Yeast Yeast cells are similar to animal cells in their core biochemistry and organelles. We should not be surprised that the 6000 yeast genes include orthologues of insulin-like signaling and other genes in tissue-grade animals (Fig. 1.3A). Fungal genomes diverged from the animals about 1500 million years ago (Cai, 2006). Aging and life span in yeast are studied with two very different experimental models: replicative life span (Piper, 2006; Sinclair et al, 1998) and chronological life span (Fabrizio and Longo, 2003; Fabrizio et al., 2004). The yeast replicative life span is defined by asexual reproduction through the formation of smaller buds on the surface of the mother cell. The intervals between budding lengthen as the replicative life span is approached, at about
  • Inflammation and Oxidation in Aging and Chronic Diseases 35 20 cell divisions. Oxidatively damaged proteins are retained asymmetrically by the mother cell (Aguilaniu et al, 2003), which may be how the detached buds start the replicative clock at zero, independent of mother cell age. The replica- tive life span model resembles the Hayflick model in that both show limited cloning. The sterile postreplicative cells may have considerable remaining life span (V. Longo, personal comm). Mechanisms in replicative aging include a unique genomic instability in ribosomal DNA (rDNA) cistrons, through aberrant recombination that causes the accumulation of extra-chromosomal rDNA circles. The rDNA instability is modulated by chromatin condensation under the control of Sir2 (silent information regulator), a NAD-dependent histone deacetylase. Increased Sir2 inhibits the aberrant recombination and extends the replicative life span. Sirtuins and their orthologues have many other metabolic activities in ani- mals (Chapter 3 and 5); e.g., diet restriction activates Sirt1 and modulates lipoly- sis in mammalian fat (Wolf, 2006). The chronological life span is defined as the cell viability during prolonged periods with limited external nutrients. Yeast and other autotrophic fungi have evolved adaptive mechanisms in their natural habitats for surviving extended periods of starvation, pending episodes of surfeit. When switched from growth media to water, yeast cells become hypometabolic, extending their life span several fold to 15–20 days. Mutations in the kinase Sch9 increase life span by increasing stress resistance and glycogen reserves. Sch9, a functional homologue of Akt/PBK, which modulates life span in animals (Fig. 1.3A), also synergizes with Sir2 (Longo and Kennedy, 2006). Ongoing studies point to the convergence of mechanisms in these seemingly different models, by the shared dependence of replicative and chronological life spans on Sch9, Ras/cyr/PKA, and Tor pathways (Longo and Kennedy, 2006). The formation of rDNA circles may be regarded as a ‘yeast disease of aging’ specific to the replicative senescence mode. Besides these single cell models, yeast can also grow as filaments (pseudohyphae) (Gognies et al, 2006), which enable the invasion of ripe fruit. Dense fungal mats can form, possibly including domains with metabolic gradients. These alternate life history modes with complex mor- phology have not been studied for aging processes. 1.2.6. The Biochemistry of Aging Aging increases the load of oxidative damage in DNA, lipids, and proteins, yeast to humans (Sohal and Wendruch, 1996; Finch, 1990). Free radicals (ROS, reactive oxygen species) generated by mitochondria are a major source of oxidative ‘bystander’ damage. Other damage comes from extracellular ROS generated by macrophages, as is prominent in atheromas. Additionally, DNA, lipids, and pro- teins become glycated in an oxidizing process that is chemically driven by glu- cose and other sugars in tissue fluids. These advanced glycation endproducts (AGEs), while not initiated by free radicals, can generate ROS in further complex reactions and by activating macrophages (RAGE pathway, receptor of AGE),
  • discussed below. In the following discussions of adverse effects of ROS, we must be mindful that ROS are essential in functions of the brain, heart, and many other organs that employ ROS in signaling processes. As examples from this large field, in the brain superoxide modulates synaptic plasticity (Hu et al, 2006), whereas in the heart nitric oxide modulates contractility (Massion et al, 2005). Intracellular ROS is mainly derived from normal mitochondrial respiration (Barja, 2004; Wallace, 2005). The respiratory chain releases electrons that form the superoxide anion (O2 ·− ) by single electron reduction of O2 (Fig. 1.11). Enzymes of free radical homeostasis include catalase; two types of supraoxide dismutase (SOD)—Cu/ZnSOD and MnSOD; and glutathione peroxidase. Superoxide is enzymatically converted by superoxide dismutase (SOD) into H2 O2 , which is then catalytically degraded by transition metals to the highly reac- tive hydroxyl radicals. These reactions are limited by the enzymatic degradation of H2 O2 by catalase, or by glutathione peroxidase. H2 O2 diffuses freely across cell membranes, unlike superoxide. 36 The Biology of Human Longevity Mitochondrial electron transport chain, Lipooxygenase, Cycloxygenase Cytochrome P450s, Xanthine oxidase NAD(P)H oxidase Uncoupled eNOS Xanthine oxidase Glucose oxidase NO2 − O2 O2O2O2 HO NO2 NO2 − NO NO HO− Fe3+ H2O2 ONOO− BH4 2H2O Enzymes-Fe3+ (MPO, CAT etc.) Fe2+ CAT, Gpx ThiolsSOD + + + + + + 2H2O Compound I (E-Fe5+) 3-NO2-Tyr Tyr 2.0 ϫ 109 6.7 ϫ 109 2e− 1e− eNOS 2x−− FIGURE 1.11 Free radicals and anti-oxidant homeostasis. Molecular oxygen (O2 ) is reduced by loss of electrons to form superoxide (O2 -•, 1e) or hydrogen peroxide (H2 O2 , 2e-). Superoxide spontaneously reacts with nitric oxide (NO). Most H2 O2 forms spontaneously, or from the dismutation of O2 -• by SOD (superoxide dismutase) and is used in cell signaling. H2 O2 is degraded by intracellular catalase (CAT), extracellular glutathione peroxidase (Gpx) or thiols. Adapted from (Cai, 2005)
  • Pathways of hydrogen peroxide metabolism. Molecular oxygen (O2 ) is reduced by loss of electrons to form superoxide (O2 − , 1e− ) or hydrogen perox- ide (H2 O2 , 2e− ). Superoxide spontaneously reacts with nitric oxide (NO) to form peroxynitrite radicals (ONOO− ). Most H2 O2 forms spontaneously, or from the dismutation of O2 − by SOD and is used in cell signaling. H2 O2 is degraded by intracellular catalase (CAT), extracellular glutathione peroxidase (Gpx), or thiols. (Adapted from Cai, 2005.) ROS are strongly associated with mitochondrial DNA damage (deletions, rearrangements, and point mutations). The age-related increase of damaged mito- chondria DNA (Wallace, 2005; Chomyn and Attardi, 2003) has become a center- piece in the molecular pathophysiology of aging (Brookes et al, 1998; deGrey, 2005; Harper et al, 2004; Van Remmen and Richardson, 2001). Mitochondrial dys- functions are found in many disorders of aging, e.g., Alzheimer disease, athero- sclerosis, atrial fibrillation, diabetes, deafness, muscle atrophy, retinal degeneration. However, cause and effect are not well resolved in these long-term processes of cell degeneration. Mitochondrial production of ROS increases with age in rat liver and muscle (Bevilacqua et al, 2005; Hagopian et al, 2005; Harper et al, 2004). ‘Proton leak’ across the inner mitochondrial membrane regulates mitochondrial ROS production with high sensitivity and increases during aging (Brookes et al, 1998; Brookes, 2004; Hagopian et al, 2005; Harper et al, 2004). Mitochondrial oxidative damage to DNA and proteins is often attributed to endogenously generated mitochondrial ROS. Because proton leak increases with oxidative damage, progressive mitochondrial impairments of various types may arise during aging through subcellular bystander damage, which propagates cell oxidative damage (Brookes et al, 1998; deGrey, 2005; Harper et al, 2004). According to the oxidant stress theory of aging, life span should be influenced by levels of enzymes or anti-oxidants that produce or remove free radicals (ROS, NOS) (Bokov et al, 2004; Sohal and Weindruch, 1996; Stuart and Brown, 2005). The role of ROS is being tested in transgenic flies and mice by varying the levels of catalase and SOD that remove ROS (Landis and Tower, 2005; Mele et al, 2006). In flies, transgenic overexpression of mitochondrial Cu/ZnSOD increased life span by >35%, while catalase overexpression did not increase life span, reviewed by Landis and Tower (2005). Mice with partial deficits of MnSOD (heterozygote knockout, Sod2+ /− ) lived slightly longer (Van Remmen et al, 2003). Although the 2.5% difference was not statistically significant, the survival curves show little overlap. This careful study also showed that SOD2 deficiency increased DNA oxidative damage (8-OH dG) and tumor incidence several-fold—e.g., lym- phomas 61% versus 22%. The lack of SOD2 deficiency on skin collagen glyco- oxidation is discussed below and in Section 1.4.2. From these results and more systematic species comparisons (Kapahi et al, 1991), I suggest that anti-oxidant mechanisms may be related to the levels of molecular turnover and repair. Flies may show these stronger effects on the life span than rodents because adult flies have no somatic cell replacement and, probably, less protein turnover, which, in mammals, removes oxidative damaged Inflammation and Oxidation in Aging and Chronic Diseases 37
  • molecules. Long-lived organisms may have needed to evolve more effective repair processes (see Section 1.2.8). Transgenic overexpression of catalase in mitochondria (mCAT) in mice increases life span by 20% (5 months) and delays important pathology (Schriner et al, 2005). This study is exemplary for its genetic design, detailed histopathology, and animal care (husbandry), even reporting the infection rate in sentinel mice. Mortality accelerations were right-shifted by increased mCAT, but without change in slope, implying that aging was delayed. Tissue changes are consistent with the Gompertz interpretation that aging is delayed. At middle age, cardiac pathology was decreased (fibrosis, calcification, arteriolosclerosis), which are common causes of congestive heart failure in human aging. In skele- tal muscle, DNA oxidation (8-OHdG) and mitochondrial deletions were decreased. These findings directly link decreased mitochondrial ROS to heart pathology, which is recognized as of inflammatory origin (Query II). In a mouse model of accelerated atherosclerosis (apoE-knockout, apoE−/− with extreme hypercholesterolemia on standard diets), the systemic overexpression of catalase decreased aortic atherosclerosis (lesion area) by 66% and decreased F2-isoprostanes (lipid oxidation product) in plasma by 45% (Yang et al, 2004). Aortic lesion size correlated strongly with aortic isoprostane levels, again con- sistent with the importance of oxidized damage in inflammation. Cu/Zn-SOD had smaller effects on aortic lesions or lipid oxidation, specifically implicating hydrogen peroxide. The hyperglycemia of diabetes is associated with another source of oxidative damage through glycation, which has not been well integrated into the free rad- ical theory of aging. Glucose and other reducing sugars can oxidize and cross- link proteins by spontaneous and complex chemical reactions with lysine and arginine sidegroups yielding ‘advanced glycation endproducts’ (AGEs) that include highly reactive carbonyls (ketones and aldehydes) (Biemel et al, 2002; Monnier et al, 2005; Stadtman and Levine, 2003). Carbonyls also form by many other free radical reactions (Stadtman and Levine, 2003). Other targets of glyca- tion are lipids (ALE, advanced lipid endproducts) (Baynes, 2003) and DNA (Bucala et al, 1984). AGE adducts accumulate progressively during aging in extracellular matrix proteins as cross-links that reduce vascular and skin elasticity (Hamlin and Kohn, 1971; Monnier et al, 2005). Aortic stiffening causes progressive increases in sys- tolic blood pressure (Fig. 1.6B) and pulse wave velocity (De Angelis et al, 2004) that are underway early in adult life. The formation of atheromas is superim- posed on these slow arterial aging processes. Diabetes accelerates these vascu- lar and lens changes, implying the importance of glucose and other blood sugars in damage to long-lived proteins during aging (Cerami, 1985). Conversely, AGE formation is slowed by diet restriction, which lowers blood glucose (Chapter 3). The lack of SOD2 deficiency on skin collagen glyco-oxidation (carboxymethyl lysine and pentosidine) in the study of van Remmen et al. (2003), discussed 38 The Biology of Human Longevity
  • previously, points to the role of blood glucose, rather than extracellular ROS in glyco-oxidation (see below). As discussed below, AGE adducts participate in oxidative stress and inflammation. Pentosidine was the first chemically characterized AGE cross-link identified in tissues (Sell and Monnier, 1989). Skin collagen pentosidine accumulates progres- sively during aging in many species, and the accumulations are accelerated by hyperglycemia and diabetes (Sell and Monnier, 1990). However, pentosidine accumulations are dwarfed by glucosepane, a recently characterized AGE that is 50-fold higher than pentosidine in skin collagen (Biemel et al, 2002; Monnier et al, 2005; Sell et al, 2005). Over the life span, glucosepane is added to about 1% of skin collagen arginine and lysine residues. This is equivalent to cross-link- ing of every five collagen molecules of normal individuals and every other col- lagen molecule in diabetics. Lens proteins accumulate far less glucosepane than skin collagen (Biemel et al, 2002). We do not yet know the specific contribution of glucosepane and diverse minor glycation products to cross-linking in skin and vascular stiffening. Besides pentosidine and glucosepane, more than 20 other adducts derive from glucose, pentose, and ascorbate. AGEs form readily in test-tube reactions of proteins with glucose or other reducing sugars through Amadori and Maillard chemistry. The resulting brownish, autofluorescent mixtures are models for brunescent cataracts and other in vivo sites of AGEs (Cerami, 1985; Monnier et al, 2005). The aorta also accumulates fluorescent AGEs (Fig. 1.6D). Oxygen levels are critical to AGE chemistry and may degrade Amadori products (Ahmed, 1986). Glucosepane formation, however, forms directly from reactions that do not depend on oxygen, and is influenced by competing reactions; e.g., in the lens, the lower glucosepane may be due to high levels of methylglyoxal (Sell et al, 2005). Tissues also differ in enzymatic removal of AGEs (deglycating amidori- ases) (Brown et al, 2005). Of critical importance to inflammation, AGE adducts activate scavenger recep- tors ‘RAGE’ (receptors for AGE) on macrophages and many other cells that stim- ulate the production of ROS via NAD(P)H oxidases (gp91phox et al.) and electron transport (Fan and Watanabe, 2003; Schmitt et al, 2006). RAGEs are also activated by the amyloid β-peptide of Alzheimer disease and by AGEs present in cooked foods (Lin, 2003; Uribarri et al, 2003) (Chapter 2). AGEs and RAGEs appear to mediate feed-forward loops of oxidative stress and inflammation that increase bystander molecular damage in atherosclerosis, Alzheimer, and other chronic inflammatory diseases (Lu et al, 2004; Ramasamy et al, 2005) (Queries II and III). RAGE activation also releases cytokines (e.g., IL-6 ) and leukocyte adhesion fac- tors (e.g., MCP-1 and VCAM-1). Feedback loops induce RAGE by TNFα through production of ROS, mediated by NFkappaB (Mukherjee et al, 2005). RAGE signal- ing pathways utilize familiar workhorses in inflammation and oxidative stress, including the transcription factor NFkappaB and PI3K (Dukic-Stefanovic et al, Inflammation and Oxidation in Aging and Chronic Diseases 39
  • 2003; Xu and Kyriakis, 2003). Moreover, PI3K interfaces with other signaling sys- tems implicated in longevity (Fig. 1.3A). Lastly, RAGE activation may stimulate feed-forward ‘vicious cycles’ by autoinduction in the same cell (Basta et al, 2005; Feng et al, 2005; Wautier et al, 2001). RAGE-dependent processes are a major focus in atherosclerosis, particularly inflammation of arterial endothelia by AGE during diabetes (Feng et al, 2005; Naka et al, 2004; Ramasamy et al, 2005) (Section 1.5.1). RAGE-dependent processes are also implicated in Alzheimer dis- ease and cancer. These observations are consistent with Query II that inflamma- tion causes further bystander damage and Query III that nutrition influences bystander damage by AGE production from hyperglycemia and by AGE present in cooked food. The molecular life span (turnover or half-life, t1/2 ) is a major determinant of accumulated damage, as exemplified by AGE accumulation in arterial elastin (Fig. 1.6D). In arteries and lungs, elastin may be almost as old as the individual, as evaluated by two independent measures: D-aspartate (Powell et al, 1992; Shapiro et al, 1991), which accumulates linearly through spontaneous racemization (Fig. 1.6D) (Helfman and Bada, 1975) and by ‘bomb-pulse’ 14 C radiolabeling1 (Shapiro et al, 1991). Human aortic elastin and cartilage collagen have t1/2 >100 y, while skin collagen is 15 y. With lab tracer labeling, rodent elastin has t1/2 of months to years (references in Martyn et al, 1995; Shapiro et al, 1991). Elastin pro- gressively accumulates glyco-oxidation (AGE) (Fig. 1.6D), at the same rate as col- lagen, when corrected for turnover (Verzijl et al, 2000). Damage to arterial elastin and collagen contributes to the loss of elasticity and stiffening that cause the increase of blood pressure during aging (Fig. 1.6B) (Section 1.6.3, below). In Alzheimer disease, senile plaque amyloid and neurofibrillary tangles also include very long-lived proteins (also bomb-pulse 14 C) (Lovell et al, 2002). Other very long- lived proteins accumulate D-aspartate in tooth dentine, eye lens, and in brain white matter myelin. The accumulating oxidative damage to these life-long molecules is associated with creeping dysfunctions in arteries, skin, and eye lens; the role in myelin dysfunction is not known. In contrast, molecules with short life spans of days to weeks have less oxida- tive damage. Diabetics accumulate glycated hemoglobin A1c, for example, which turns over at the erythrocyte t1/2 of about 120 d. Erythrocyte turnover scales with body size across species (M0.18 ) (Finch, 1990, p. 289). The t1/2 of many proteins is allometrically related to body size and may be a crucial determinant of the rates of damage accumulated during aging across species. Moreover, the rates of basal metabolism correlate with molecular turnover in species comparisons of mammals. The insulin-like signaling pathways (Fig. 1.3) may mediate many of these fundamental energy relationships. 40 The Biology of Human Longevity 1 An unplanned 14 C tracer labeling event occurred from atmospheric testing of nuclear weapons in the early 1960s; environmental 14 C has now returned to pre-1955 levels (Lovell et al, 2002).
  • Aging slows the turnover of many shorter-lived proteins (Finch, 1990, pp. 370–373; Goto et al, 2001)—e.g., bulk proteins in worm (Reznick and Gershor, 1979) and in mouse liver (Lavie et al, 1982; Reznick et al, 1981). The causes of slowed turnover during aging are not known and could include impaired proteasomal degradation, as in aging rodents (Goto et al, 2001). These metabolic level aging processes thus tend to accelerate the accumulation of oxidized damage. The effectiveness of diet restriction in slowing aging may be due in part to the accelerated protein turnover and decreased oxidative load (Chapter 3). The balance of reduction:oxidation (‘redox’) in glutathione and other key homeostatic regulators (Fig. 1.11) is shifted to a more oxidized state (GSSG and protein-SSG) in blood, liver, and other tissues (Lang et al, 1989; Lang et al, 1990; Rebrin et al, 2003; Rebrin and Sohal, 2004), and in whole aging flies (Rebrin et al, 2004). Glutathione, the major redox couple, is at much higher levels than other redox links involving cysteine, thioredoxin, NAD, etc. (Sies, 1999). In healthy aging humans, blood GSH remains relatively stable; however, with cardiovascu- lar disease, diabetes, or kidney disorders, blood GSH tends to drop below the normal range (Lang et al, 2000). Similar redox shifts occur during chronic infec- tions; e.g., in HIV patients, blood GSH decreases in proportion to the viral load (Sbrana et al, 2004). Conversely, redox shifts are opposed by diet restriction (Chapter 3, Fig. 3.14). A component of the GSH shifts of aging could also be a response to low-grade infections, which would also be consistent with the increase of CRP, IL-6, and other acute phase reactants in aging populations (see below and Chapter 2). It is important to resolve the contributions to the oxidized load of aging from three sources: (I) endogenous mitochondrial free radicals and other tissue processes; (II) interactions with the commensal gut and skin flora; and (III) specific infections. The naked mole rat (Heterocephalus glaber) is adding surprising findings to these debates. H. glaber is the most longevous rodent (at least 28 y), yet has sim- ilar body weights of lab mice (30–80 g) (Andziak et al., 2006; Andziak and Buffenstein, 2006). In comparison with lab mice (CB6F1) at 10% of the lifespan (24 m vs. 4 m), H. glaber had indicators of greater oxidative stress than in lab mice, e.g., 10-fold more urinary isoprostanes, 35% higher myocardial iso- prostanes, and 25% lower hepatic GSH:GSSG ratios. While it might be concluded that sustained oxidative stress is not incompatable with extraordinary longevity, much remains to be learned about other aspects of metabolism in these remark- able animals. Its membrane lipids differ from other mammals by much lower levels of unsaturated fatty acids in muscle and brain, particularly docosa- hexaenoic acid (DHA or 22:6 n-3), which is highly susceptible to peroxidation (Hurlbert et al., 2006). The oxidizability of membranes (peroxidation index) fits well with the inverse allometry on lifespan. These varying results across species suggest that anti-oxidant mechanisms vary within and between phyla. Besides differences in membrane composition and anti- oxidants, I suggest the importance of molecular turnover and repair. Flies may show Inflammation and Oxidation in Aging and Chronic Diseases 41
  • these stronger oxidative effects on the life span than rodents because adult flies have no somatic cell replacement, except in the gut, and, probably, less protein turnover, while mammals have extensive molecular turnover, which removes oxida- tive damaged molecules. Long-lived organisms may have needed to evolve more effective repair processes (see Section 1.2.8). 1.2.7. Biomarkers of Aging and Mortality Risk Markers For populations, the life span is expressed statistically, often as the life expectancy. However, no measurement has been found that accurately predicts the individual life span from the genotype, or from any ‘biomarker of aging.’ Clearly, the traditional aging changes of gray hair and menopause do not assess an individual’s current health or future longevity. Jeanne Calment lived 70 years after menopause to achieve her longevity record of 122 years. The N.I.A. has supported an extensive search for biomarkers that predict remaining life span (Biomarkers of Aging Program, begun in 1982) (Reff and Schneider, 1982). Biomarkers have been evaluated in biochemical, cellular, genetics, molecular, and physiological characteristics, and in behavior and cog- nition. Emphasis has been given to changes of ‘non-pathological aging’ that are distinct from disease. Two decades later, no single biomarker or combination has been found to predict longevity better than the individual age in fly, worm, rodent, or human (Finch, 1990, pp. 558–564; Warner, 2004). Consider the limits of biomarkers in aging worms, which seem an optimal model for actuarial questions by their minimal genetic, environmental, and social heterogeneity from dominance hierarchies and social interactions (worms are self-fertilizing). Pharyngeal pumping, by which food is ingested, declines progressively during the first week and then nearly ceases a few days before death. Body movements closely parallel the pumping rates, not surprisingly because pumping provides the food needed for energy. The duration of fast pharyngeal pumping and body movements shows strong correlation with indi- vidual life spans (Huang and Kaley, 2004). When fast pumping is maintained one day longer, the odds ratio for death by, or later than, a specified date is 1.7- fold greater. The Spearman rank correlation coefficient for the duration of fast pumping and remaining life span was highly significant (r 0.49, P< 0.0001). Despite this statistic, the fraction of life span variance explained by pumping or movement was only 24%. Thus, other variables besides pumping account for about 75% of the individual differences in life span. Mutants with greater longevity have longer phases of active body movement and pharyngeal pump- ing. (Section 5.5.2). Even at hatching, worms differ hugely in movements, which Kirkwood and I attributed to chance variations in cell organization and gene expression during development (Finch and Kirkwood, 2000, pp. 58–65). A concrete example is Tom Johnson’s elegant study of worm-to-worm variations in expression of a 42 The Biology of Human Longevity
  • stress-protective gene (hsp-16.2) in young worms, which correlated strongly with individual life spans (Rea et al, 2005). Again, the Spearman coefficient of 0.48 accounts for only 25% of the variance in life span. These sobering examples from rigorous studies suggest limits in the predictability of life spans despite strictly controlled genetics and environment. In worms, as in flies, mice, and humans, the heritability of the life span is also about 25% (Finch and Tanzi, 1997; Finch and Ruvkun, 2000) (Section 5.2). As noted above, partial deficits of MnSOD did not alter mouse life span (Van Remmen et al, 2003). Several biomarkers of aging that respond to diet restriction were the same in Sod2+/− as in control aging mice (ad libitum fed in this study): both genotypes had identical age-related decreases of spleenocyte proliferation and increases in skin collagen of pentosidine and carboxymethyllysine. The lack of effects of MnSOD deficiency on these AGEs indicates that blood glucose was not altered. However, Sod2+/− mice had greater accumulations of oxidized nuclear DNA (8oxodG) in brain, heart, and liver, and 2-fold more lymphomas (83% vs. 41%). This study with its careful analytical chemistry and histopathol- ogy shows the uncertainty of connections between robust biomarkers of aging, tumor prevalence, and the life span. There is reason to consider the individual disease load as more informative than tissue-level aging changes in predicting mortality risk (Karasik et al, 2005). During these same decades of the Biomarkers Program, vast clinical research has developed risk indicators of mortality for the major diseases. From the clin- ical perspective, disease, not aging, is the cause of mortality, as shown in the exponential increase of tissue lesions in rodent models (Fig. 1.5) and heart attack and stroke in human populations (Fig. 1.6C). Systolic blood pressure may be the most robust overall indicator of human mortality risk, with exponential age-related increases of future heart attack and stroke at all levels of systolic pressure. The next phase of the biomarker debate may redefine the often vaguely used term ‘disease.’ For example, the clinical threshold of hypertension as a target for intervention is being expanded to include the ‘high normal’ range. A few decades ago, an informal guideline of the expected systolic pressure was “add 100 to the person’s age”! Combinations of risk indicators and disease load are also being intensively studied of the inflammatory marker, C-reactive protein (CRP). In the Women’s Health Study, future heart attacks occurred most frequently in those with both elevated CRP and LDL (Ridker, 2002). New models are needed to resolve the links between the diverse subtle subclinical aging changes that inter- act to cause circulatory failure on the background of declining organ reserves. It is shocking that 30% of diet-restricted old rats had no gross lesions at necropsy and cause of death was unknown (Shimokawa et al, 1993). Declining home- ostasis of glucose and electrolytes, for example, might allow transient distur- bances that would arrest a fibrotic heart, even in the absence of thrombotic Inflammation and Oxidation in Aging and Chronic Diseases 43
  • blockade. The combinatorics of various mild dysfunctions gives rise to a huge number of individual pathophenotypes that may be each estimated as relative risk of mortality, but may never account for more of the variance in life span than in the worm model. 1.2.8. Evolutionary Theories of Aging The demographics of natural populations are the basis for evolutionary theo- ries of aging. In humans, like most other animals, the major phase of repro- duction is accomplished by the young adults. Mortality from arterial and malignant diseases is low until after age 35, which approximates the life expectancy in most human populations before the 19th century (Fig. 1.1A). High levels of extrinsic mortality allow only a minority of humans to survive to older ages, until very recently. The major early causes of mortality are due to extrinsic risks, including infections, malnutrition, and trauma. A demographic pyramid with young adult ages as the largest is widely observed in natural pop- ulations. Sufficient reproduction to maintain the population can be distributed in many combinations of age groups, all governed by the level of mortality that allows a sufficient number to survive to adult ages and to reproduce sufficient numbers of offspring, which themselves survive to become reproductive. The reproductive schedule includes the duration of maturation, when individuals are at risk for dying before reproducing, as well as the frequency of reproduc- tion and duration of the reproductive phase. The duration of the reproductive schedule is the prime determinant of lifespan (Hamilton, 1966; Austad, 1993, 1997; Rose, 1991, 2004). Populations may be described by the Euler-Lotka equation, which is based on life table calculations as the net reproductive rate averaged over age classes, x (Hamilton, 1966; Rose, 1991; Stearns and Koella, 1986). The rate of population growth (r) is calculated at each age class (x) from the sum of the products of the mortality rate m(x) and the reproductive rate b(x). x ∑ o exp(−rx)m(x)b(x) = 1. The net population growth can not be negative for very long, or extinction results. Thus, increases in mortality m(x) must be compensated by commensu- rate increases in reproduction in one or another age b(x). Note that this equa- tion is the sum of terms that are commutative products in each age, e.g., the number 8 is the product of (2 × 4) or (4 × 2) etc. Thus, an infinite number of dif- ferent combinations of m(x) and b(x) can satisfy this equation. Because life expectancies are determined by mortality rates, we may speak naturally of the lifespans in plural that characterize a species. Lifespans are highly plastic and changeable in relation to the reproductive schedule and reproductive output. Experimental tests of these relationships are described below. 44 The Biology of Human Longevity
  • The duration of lifespans can vary widely in conjunction with the reproduc- tive schedule. At one extreme are species that die after producing vast numbers of fertilized eggs, of which few survive to adulthood, such as in the five species of Pacific salmon (Finch, 1990, pp. 83–95). At the other extreme are species like the great apes that typically have one child at prolonged intervals, of up to ten years in orangutans (Wich et al, 2004). Differences in extrinsic mortality between populations may lead to different reproductive schedules. In the common opossum (Didelphis virginiana), popula- tions on isolated islands, which are exposed to low predation, are observed to mature more slowly and have fewer offspring per litter (Austad, 1993, 1997). Moreover, aging seems to be delayed, with slower mortality accelerations, slower collagen aging and longer lifespans than mainland opossums, which are under higher predation. These observations are consistent with the hypothesis that senes- cence is delayed if predation levels are lower in species comparisons (Edney and Gill, 1968), and Steve Austad’s corollary that aging should also be modified commensu- rate with the reproductive schedule as external predation varies. Human groups also vary in schedules of maturation and reproduction (Chapter 4, Fig. 4.5C). For example, in pre-industrial foragers, menarche occurs in the Ache by 14 y and first child at 17.7 y, while the Jo/’hoansi menarche is at 16.6 and first child at 18.8 (Walker et al, 2006). The precocious extreme is found in privileged populations with excellent health from abundant food and low loads of infections, where girls have much earlier menarche 12 years or less, closer to menarche in the great apes (Fig. 6.2). While we do not know the rela- tive contribution of environmental factors and genetics in these populations, twin studies show that growth rates, age at menarche, and age at menopause have significant heritability (Chapter 5.2). The force of natural selection is considered to decline during aging (Fig. 1.12A) because the majority of reproduction is achieved by the younger adults, which are the dominant age group in most natural populations due to external causes of mortality distinct from aging (Hamilton, 1966; Rose, 1991). The declining force of natural selection during aging is then considered permissive for the accumulation over time of mutations in the germ-line with delayed adverse effects emerging in adults. Examples include rare dominant familial genes for Alzheimer disease, breast cancer, or hyperlipidemia, which have little impact on young adults. Such delayed consequences of adverse mutations are not strongly selected against when effects are delayed to later ages that con- tribute less to reproduction. If a dominant gene caused earlier dysfunctions, its carriers would have difficulty competing for mates, and the early age phenotype would soon disappear. These concepts were introduced by J.B.S. Haldane (1941) and Peter Medawar (1952), and developed into rigorous theory by George Williams (1957) and William Hamilton (1966). In another hypothetical case, genes with adverse later effects might be selected by their benefit during devel- opment or in early ages—Williams’ ‘antagonistic pleiotropy’ hypothesis (Williams, 1957; Williams and Nesse, 1998). Thus, the schedule of reproduction Inflammation and Oxidation in Aging and Chronic Diseases 45
  • 46 The Biology of Human Longevity Age A Rate Aging, disease, and mortality Chronic diseases Mortality Gene-Environment Force of Natural Selection B Selection for Longevity in Drosophila 0 10 20 30 40 50 −40 −20 0 −10 −30 10 20 30 D Old-youngEggs/day 0 10 706050403020 80 %Survival D 0 100 80 60 40 20 Old Young 0 100 80 60 40 20 %Survival Y O FIGURE 1.12 Evolutionary demography. A. Natural selection in aging, disease, and mortality in organisms with senescence. The force of natural selection declines during aging because most repro- duction is accomplished by younger adults. With advancing age, the rate of chronic disease incidence increases exponentially (arterial events, cancer, Alzheimer disease), followed by increasing rate of mortality, as shown for rat in Fig. 1.5. Gene-environment interactions can shift these disease mor- tality curves, as shown for historical changes in humans (Fig. 1.1B, Fig. 2.7). Original figure (CEF). B. Using selection for reproduction at later age reproduction in flies (outbred Drosophila melanogaster) yielded flies that lived about 20% longer within 15 generations and had more gradual mortality acceleration; conversely, selection for early reproduction shortened life span and accelerated aging. The bottom panel shows differential in egg production between young and old selected lines. Redrawn from Rose (1984, 1991) and Luckinbill et al. (1984). Rose progressively increased the later age selected for reproduction, whereas Luckinbill maintained the selected ages.
  • Inflammation and Oxidation in Aging and Chronic Diseases 47 for any species to survive (maintain Darwinian fitness) sets a lower age limit in the development of adverse phenotypes. Many studies show the power of experimentally manipulating the repro- ductive schedule. In a now classic paradigm in the experimental evolution of aging, flies were selected for reproduction at later ages (Rose and Charlesworth, 1980; Rose, 1991). Within 15 generations, this selection regime yielded flies that lived about 20% longer and had more gradual mortality accel- eration (Luckinbill et al, 1984; Rose, 1984; Rose et al, 2004) (Fig. 1.12B). Conversely, selection for early age reproduction shortened life span (Fig. 1.12B). These powerful effects require outbred populations and do not depend on the spontanteous emergence of new mutations and their fixation. Rather, shifts in genotype are attributed to frequency change of existing alleles, which occurs at a vastly greater rate than the mutation frequency. Moreover, the reproductive schedule and lifespan can be shifted back toward initial values in a “reverse evolution” paradigm, by switching the ages of selection (Teotonio et al, 2002; Rose et al, 2004). These studies also show important gene-environment interactions in the density-dependence of lifespans, which were sensitive to population density in both larval and adult stages (Mueller et al, 1993; Rose et al, 2004). Density- dependent effects could involve microbial flora which grow on excreta and dead larvae. Bacteria (Hurst et al, 2000) and fungi (Rohlfs, 2005) are well known to influence larval growth and even the age of phenotype expression (Hurst et al, 2005). The microbial environment could also be important in aging fly populations that have greatly increased microbial load (Renn et al, 2007; Section 5.6.4). However, unexpected results have come from studies of the guppy (Poecilia reticulata), which experimentally varied predation pressure (Reznick et al, 2001; Reznick et al, 2006). In some localities with intense predation and with high extrinsic mortality, fish mature earlier and have higher early fecundity. These different reproductive schedules show heritability that persists in the laboratory. Just as in the fly experiments, altering the extrinsic mortality from predation pressure in wild populations caused corresponding advance or delay of reproduction. When guppies were allowed to live out their days in the lab, the early maturing fish reproduce for an additional 200 days longer than the later maturing fish, contrary to the natural populations of opossums or the lab fly studies above. Moreover, the post-reproductive life spans did not differ between the early and late reproducing lines. These intriguing results challenge the evolutionary theory of senescence that was comfortably accepted for several decades. Moreover, not all animals show reproductive senescence at advanced ages. Two species of turtles (painted, Blanding’s turtles) have not shown decline in fecundity at ages over 60 y in longitudinal field studies (Congdon et al, 2003). Some older individuals continue to be successful in laying and protecting their
  • egg clutches to hatching, as much as the average young adult. Similarly, the very long-lived rockfish (Sebastes) maintain seasonal cycles of de novo oogenesis at least up to age 80, and does not show evidence for declining fecundity (Finch, 1990, pp. 216–219; Finch, 1998; De Bruin et al., 2004). These and many other examples support the possibility of ‘negligible senes- cence,’ or negligible decline in reproduction and other functions during the nat- ural life span (Finch, 1990, pp. 207–247; Finch, 1998). ‘Negligible senescence’ as a potential life history type was considered theoretically untenable by most biogerontologists when I first proposed it, at least in part because of Hamilton’s highly regarded mathematical model, which concluded “.... even under utopian conditions [of exponential fertility increase and immortality], given genetic varia- tion....phenomena of senescence will tend to creep in” (Hamilton, 1966, p. 25). However, further theoretical developments by James Vaupel and colleagues (2004) show that the level of senescence is highly sensitive to assumptions and parameter values and that there may even be ‘negative senescence’ under some conditions. Another challenge to evolutionary theory of senescence is the stable charac- teristics of senescence in related species, whereas evolutionary theory of the weak selection against later deleterious phenotypes should yield a great variety of different aging genes and great individual variability in aging. Indeed, there is great variability in life span, and specific aging phenotypes is certainly manifest between individuals. And, as well studied in twins, life span has low heritability (Section 5.2). Nonetheless, at the species and population level, some aging changes are so generally found that they may be described as canonical patterns (Table 1.1). In all well-studied human populations, arterial elasticity decreases and systolic blood pressure increases with adult age; menopause occurs by age 55 due to depletion of ovarian oocytes; reproductive tract tumors become increasingly prevalent; and bone density decreases, while the risk of disabling fractures increases. These canonical changes of human aging also characterize aging in other mammals (Finch, 1990, pp. 154–155), including details of arterial aging (Table 1.4, Section 1.5.3.3). The basis of the canonical patterns in aging may be sought in development: Mammals share a basic body plan and program of embyronic gene regulation that determines the patterns of gene expression in differentiated cells. The human genome regulatory machinery was established at least 150 million years ago in mammalian ancestors and is apparently resistant to evolutionary change because of the densely connected transcriptional circuits required for embryonic development (regulatory kernels) (Davidson, 2006). Future studies may show the level of gene circuit regulatory lock-in that determines the replacement of mole- cules and cells in adults. As discussed above (Section 1.2.6), arterial elastin and other long-lived molecules inevitably accumulate molecular damage (Fig. 1.6D), whereas in circulating erythrocytes, the genomically programmed cell replace- ment with 120 day lifespans allows much less persistence and impact of oxida- tive damage. Thus, we must look to genomic regulation during development to 48 The Biology of Human Longevity
  • understand the cell phenotypes that underly tissue aging processes and that may also be the basis for species differences of lifespan. Molecular and cell repair, replacement, and organ regeneration require meta- bolic energy. Tom Kirkwood’s ‘disposable soma’ theory of aging recognizes that energy resources are finite (Kirkwood and Holliday, 1979; Drenos and Kirkwood, 2005). Molecular and cell repair and regeneration require metabolic energy. At each moment, the individual organism assesses its homeostatic condition and allocates energy accordingly. Insufficient food intake attenuates immunity (Chapter 3) and growth (Chapter 4). We will examine this in the impact of diet restriction, which can attenuate immune defenses (Chapter 3), while infections attenuate growth (Section 4.6) and reproduction (Section 5.4.2). Each species has evolved within a relatively predictable balance of energy availability and energy that must be allocated for reproductive success. As one of many examples, when worker bees leave the hive to forage, a highly dangerous activity that accelerates mortality (Finch, 1990, p. 70), they decrease their immune defenses (Amdam et al, 2005). This trade-off of foraging energy against immune defenses is statis- tically favored because of the ultra-short life span of field bees. Theoretical mod- eling of trade-offs between investment in somatic maintenance and fecundity (Darwinian fitness) shows very broad curves of fitness, with optima that allow considerable variation in somatic investment (Drenos and Kirkwood, 2005). These results are consistent with observations that many biological functions do not decline appreciably and that major species differences are to be expected. A future goal of aging theory is to understand the physiological trade-offs of immu- nity, repair, and regeneration that define the species reaction norm of mortality rates to environmental fluctuations (Stearns and Koella, 1986). 1.3. OUTLINE OF INFLAMMATION Inflammatory processes of innate immunity are evident participants in many tissue changes of normal aging and most of the chronic degenerative diseases of aging, as outlined in Fig. 1.2A and discussed throughout this book. Innate immune responses are the standing initial defense system against invading pathogens. The acute phase of innate immunity is mediated by secretion of sys- temic ‘acute phase’ proteins and the local activation of macrophages with rapid production of free radicals (Fig. 1.11). Acute phase responses do not depend on prior immune experience of adaptive (instructive) immunity, mediated by B- and T cells. However, exposure to new antigens during the acute phase response can activate the targeted immune responses to pathogen antigens by B- and T cells. Tissue injury from trauma or toxins can also induce inflammatory processes that go far beyond free radical production in tissue matrix remodeling and repair. Host defenses require energy, which is allocated in trade-offs, as just discussed, that determine the duration and type of inflammatory responses and the level of repair and regeneration. Inflammation and Oxidation in Aging and Chronic Diseases 49
  • 50 The Biology of Human Longevity The inflammatory processes at work in atherosclerosis, Alzheimer disease, diabetes, and obesity include a shared set of acute phase responses (Sections 1.5, 1.6, 1.7). Some of the same processes that macrophages employ to remove bacterial invaders are also implicated in arterial disease through the uptake of lipids by macrophages. Blood lipid responses to infections (lipid oxidation, elevated acute phase proteins, triglyceridemia) are also an athero- genic profile. Moreover, emerging evidence indicates roles for T cells in ath- erosclerosis (Section 1.5.3). Other tissue lesions of aging that involve chronic inflammation may also arise from an early seeding injury. Gastro-intestinal cancer, for example, is associated with local inflammation in response to H. pylori infections (Section 2.8.1). Remarkably, many of the acute phase genes are also upregulated in normal aging, in the absence of these specific lesions (Section 1.8). Cause and effect are unresolved in these complex, long-term processes. 1.3.1. Innate Defense Mechanisms The acute phase of the innate immunity (‘emergency line’ ring-ups’) include the ancient cardinal signs of inflammation in a localized injury: heat (calor), redness (rubor), swelling (tumor), and pain (dolor).2 ‘Inflammation’ is now understood to include the vastly complex, multi-organ system of defense and repair, in which free radicals have major roles (Fig. 1.11) in directed cytotoxi- city and in normal cell signaling. Acute phase reactions can be stimulated by invading pathogens within one hour and, depending on the level of activation and efficacy of initial defenses, may continue for days or longer. These critical initial defenses rapidly enhance the removal of pathogens by phagocytosis, e.g., by induction and secretion of C- reactive protein (CRP), which is bacteriocidal by binding to Gram-negative bac- teria and enhancing their removal by macrophages through phagocytosis. Blood clotting is enhanced by the increase of fibrinogen and other prothrombotic changes. Of major importance, energy resources are mobilized for increased cell activities and systemic responses such as fever. The liver rapidly secretes inflam- matory proteins designed to neutralize invading organisms and to mobilize the needed energy. The acute phase inflammatory responses are coded by ancient genes with equivalents (orthologues) in invertebrates (Section 5.4, Fig. 5.4) as 2 Cornelius Celsus (1st C. encyclopedist) in De Medicina described inflammation as “rubor et tumor cum calore et dolore” (redness and swelling with heat and pain), later expanded by Galen (2nd C. physician) “ ...et functio laesa” (loss of function) (Plytycz and Seljelid, 2003). The inflammation in atherosclerosis and Alzheimer disease does not cause pain because the affected tissues are not innervated by pain fibers; however, joint pain from osteoarthritis is all too familiar during aging.
  • well as in lower vertebrates (Azumi et al., 2003) and plants (Ezekowitz and Hoffman, 2003). Transcriptional regulation is at the core of inflammatory responses, often mediated by NF-kB and PPAR. The acute phase of inflammatory responses may be followed within days by the adaptive immune responses of lymphocyte B-cells and T-cells that recognize new antigens on an infectious invader. Antigen-stimulated immune responses involving somatic gene recombination occur in vertebrates, from bony fish to mammals, but were not evolved in worms, flies, and other invertebrates (Azumi et al, 2003; Marchalonis et al, 2002). Inflammation in ‘normal’ aging involves the same cells and molecules found in the pathology of arterial atheromas and senile plaques (Table 1.3). I focus on CRP and certain interleukins (IL-1␣, IL-6, IL-8, IL-10). These and other acute Inflammation and Oxidation in Aging and Chronic Diseases 51 TABLE 1.3 Inflammatory Components of Vascular Atheromas and Senile Plaque Atheroma Senile Plaque cells astrocytes 0 + + mononuclear cells macrophage + + + (foam cell, macrophage; CD68) + + (microglia, CD68) T-cell + + (CD3 CD4/Th1) 0 mast cells + + 0 platelets + + 0 neovascularization + + 0 proteins amyloids Aβ ? (macrophages with ingested platelets) + + CRP + + + (neurites) SAP + + clotting factors + + 0 complement C5b-9 + (fibrinogen) + cytokines: IL-1, -6 + (associated with CRP) + metals: Fe Cu, Fe, Zn abbreviations: 0, absent; +/−, weak; +, definitive; + +, moderate; + + +, extensive. amyloids, Aβ: atheroma (De Meyer et al, 2002); senile plaque (Glenner and Wong, 1984; Hardy and Selkoe, 2002; Klein et al, 2001); CRP : atheroma (Rolph et al, 2002; Torzewski et al, 1998); senile plaque (Akiyama et al, 2000; Veerhuis et al, 2003); SAP: atheroma (Li et al, 1995; Meek et al, 1994); senile plaque, (Coria et al, 1988; Veerhuis et al, 2003). Aβ is detected in carotid artery plaque macrophages (De Meyer et al, 2002) and could be derived from platelets adherent to plaques, which contain the amyloid precursor protein (APP) and, when activated, release APP and Aβ-containing peptides (Jans et al, 2004); platelet APP-derived protease nexin 2 (PN-2) is an anti-coagulant (Van Nostrand, 1992). C5b-9 (membrane attack complex); SAA, serum amyloid A (Akiyama et al, 2000; Finch, 2002); B cells: atheromas (Millonig et al, 2002); T-cells: atheromas (Benagiano et al, 2003; de Boer et al, 2006; Häkkinen et al, 2000; (Millonig et al, 2002); mast cells: atheroma (Kelley et al, 2000; Millonig et al, 2002); senile plaque platelets: atheroma (De Meyer et al, 2002; Nassar et al, 2003; von Hundelshausen et al, 2001); complement: atheroma (Torzewski et al, 1998); senile plaque (Akiyama et al, 2000; Eikelenboom and Stam, 1982; McGeer et al, 1989); cytokines: atheroma (Rus et al, 1996); senile plaque (Akiyama et al, 2000). metals: atheromas with intraplaque hemorrhages that deposit extracellular iron; also iron in macrophage from erythrocyte debris (Kolodgie et al, 2003); senile plaques accumulate metals in plaque core and periphery, and colocalized with amyloid fibrils (Lovell et al, 1998; Miller et al, 2006).
  • 52 The Biology of Human Longevity phase proteins are secreted by the liver (Bowman, 1993), but also by macrophages and other cells elsewhere. The amyloid precursor protein (APP) may also be an acute phase response in the brain (Section 1.6.2). These exam- ples illustrate, but cannot fully represent the remarkable pleiotropies observed in the hundreds of inflammatory mediators. When pathogens enter an organ, host defense systems respond immediately to isolate or destroy the invader and to close wounds to prevent further invasion from the skin, airways, or digestive tract. Pathogens are removed by ‘professional’ phagocytes, the ancient macrophage cells residing in every tissue and in the cir- culation. Acute phase responses are stimulated by receptors that recognize ‘pathogen-associated molecular patterns’ (PAMPS) of common invaders hypothe- sized by Janeway (Dalpe and Heeg, 2002; Medzhitov and Janeway, 2002; Netea et al, 2004). Bacterial PAMPs include their outer coat components, known as the ‘endotoxins’: lipopolysaccharide (LPS) of gram-negative bacteria (e.g., Escherichia coli) and the lipotechoic acid (LTA) of gram-positive bacteria (e.g., Staphylococcus aureus). LPS is recognized by specialized receptors in many cell types. In liver cells, LPS activates the Toll-4 receptors, leading to rapid secretion of CRP, IL-6, and TNFα. Bacteria are also inactivated by binding to blood lipoproteins (low- and high-density lipoproteins, LDL and HDL), which have specificities for LPS and other bacterial coat components (Khovidhunkit et al, 2004). HDL and LDL, for example, bind LPS. Some viruses are also inactivated by lipoproteins, e.g., Epstein-Barr, Herpes simplex virus. These and other pathogens are also implicated in vascular disease (Chapter 2). Foci of chronic inflammation are broadly associated with cell proliferation. Hyperproliferation is hypothesized to be the cause of mutations arising through errors in DNA synthesis that increase cancer risk (bystander damage, Section 1.4). This hypothesis is well developed for the role of Helicobacter pylori in intestinal cancer (Section 2.9) and is being considered for other epithelial cancers, e.g., bladder and endometrium (Dobrovolskaia and Kozlov, 2005; Modugno et al, 2005). Tissue fibrosis is also broadly associated with inflammation and may be con- sidered as generalized wound healing response, which increases fibroblast prolif- eration and deposition of extracellular collagen and other matrix material. For example, myocarditis from Coxsackie virus infections developed focal scars with thickened collagen networks around surviving myocytes (Leslie et al, 1990). Interstitial myocardial fibrosis also arises during aging in the absence of defined infections in rodents and humans (discussed in Section 1.2.2), and is associated with increased myocardial stiffness during aging (decreased ‘compliance’) (Brooks and Conrad, 2000; Lakatta and Levy, 2003a,b; Meyer et al., 2006). Fibrosis is very common during mammalian aging and deeply linked, if not intrinsic, to general inflammatory processes in aging (Thomas et al, 1992). TGF-β1 signaling pathways regulate collagen synthesis and are implicated in fibrosis of liver (Lieber, 2004), lung (Chapman, 2004), and myocardium (Brooks and Conrad, 2000; Sun and Weber, 2005).
  • Many inflammatory mediators also have normal functions. (Gene classification systems that may be helpful in organizing data on expression should not be regarded as exclusionary of functions.) IL-6 illustrates these pleiotropies, which range from local cell effects to behaviors of the whole organism. While most circulating IL-6 is secreted by the liver, IL-6 is also made by adipocytes, neurons, and many other cells. IL-6 expression is regulated by transcription factors of con- vergent pathways that integrate local tissue and systemic signals (Kubaszek et al, 2003; Trevilatto et al, 2003). Moreover, IL-6 and CRP mutually stimulate tran- scription of the other genes (Arnaud et al, 2005). At a local site of injury, IL-6 regulates cell adhesion molecules that capture circulating neutrophils and macrophages (Kaplanski et al, 2003; Nathan, 2002). IL-6 induction can protect local cells from free-radical-induced death (Waxman et al, 2003). IL-6 is a stimu- lator (growth and differentiation factor) of B- and T-lymphocytes (Ishihara and Hirano, 2002; Zou and Tam, 2002). IL-6 also influences metabolism and stimu- lates the resting metabolic rate (BMR); the linear dose response to IL-6 increases BMR by up to 25%, as observed during fever (Tsigos et al, 1997) (Fig. 1.2B). Sustained elevations of IL-6 induce fever-related behaviors of lethargy and poor appetite. Thus, IL-6 sits in a highly integrated network sensitive to the energy state of the individual. After the acute phase is initiated, a slower phase of adaptive immune responses may be initiated in which lymphocytes are mobilized to recognize new antigens. The differentiation and proliferation of new lymphocyte clones is regulated by IL-1, IL-6, and other interleukins (historically known as lymphokines). The course of inflammatory responses is regulated by many checks and bal- ances (Li et al, 2005; Nathan et al, 2002; Tracey, 2002) besides the anti-oxidant systems (Fig. 1.11). Local activation of the complement system (C-system) cascade is checked by inhibitors and short molecular life spans (‘tick-over’) of activated C-complexes (Morgan, 1990; Rother and Till, 1988). The complement system is integrated with inflammatory responses and is regulated by cytokines. Among many examples, TGF-ß1 represses the first component of the classical C-pathway, C1q mRNA (Morgan et al, 2000). To counter the damage from the ongoing production of free radicals, body fluids and cells have strong anti-oxidant mechanisms. The redox recycling of glu- tathione in cells and in the blood is very important. Other regulators also inhibit or modulate inflammatory responses; e.g., local TNFα release may be inhibited by so-called anti-inflammatory cytokines, which include TGF-␤1 and IL-10 (Elenkov and Chrousos, 2002). Free radicals generated by inflammation also induce DNA repair mechanisms. For example, gastric infections of Helicobacter pylori cause infiltrations of immune cells that generate free radicals, in turn, increasing oxidative DNA damage and apoptosis in the gastric mucosa. Experimentally, H. pylori and H2 O2 induce enzymes that repair oxidative DNA damage—e.g., the redox-sensitive transcrip- tion of APE-1/Ref-1 (apurinic/apyrimidinic endonuclease-1/redox factor-1), a multi-functional enzyme that mediates base excision repair of oxidatively Inflammation and Oxidation in Aging and Chronic Diseases 53
  • damaged DNA (apurinic sites) (Ding et al, 2004; Lam et al, 2006). This adaptive response (one of many) increases cell resistance to genotoxic free radicals (Ramana et al, 1998). The immune system is hormonally integrated. Pituitary growth hormone (GH) induces hepatic production of insulin-like growth factor (IGF), which is a medi- ator of immune and inflammatory functions (Denley et al, 2005; Russo et al, 2005). IGF-1 also feeds back to inhibit pituitary GH secretion. IGF-1 signaling utilizes the PI3 kinases (phosphatidylinositol 3-kinase isoforms) that are general workhorses in signaling (Fig. 1.3A, B). Macrophages have high-affinity IGF-1 receptors and also secrete IGF-1 (Bayes- Genis et al, 2000), which may be important to both atherogenesis and Alzheimer disease (Section 1.6.4). IGF-1, but not GH, stimulates macrophage secretion of TNFα (Renier et al, 1996). Sex steroids influence immune functions response to trauma (Angele et al, 2000) and pathogens (Soucy et al, 2005). The deep associ- ations of reproduction and immunity enable the evolutionary optimization in the face of the endless assault by infectious pathogenic organisms. Evidence that this selection is ongoing is the extensive genetic variations found in inflammatory responses. 1.3.2. Genetic Variations of Inflammatory Responses Many genes that influence inflammatory responses may influence the risk of and course of vascular and Alzheimer disease (Chapter 5). Identical twins show extensive heritability in cytokine responses to LPS, accounting for a striking 50% or more of the variance in IL-1β, IL-6, IL-10, and TNFα (de Craen et al, 2005). The IL-6, IL-10, CRP, and apoE gene variants in regulatory and coding elements influence responses to infections (Bennermo et al, 2004; Kelberman et al, 2004). In bacterial meningococcal meningitis, children carrying an IL-6 promoter vari- ant with lower IL-6 production had 2-fold better survival (Balding et al, 2003). The lower production of IL-6 by the meningococcal LPS may reduce local bleeding (microvascular thromboses). Similarly, gene variants of IL-1β influence inflamma- tory responses to infections by Helicobacter pylori, a common pathogen, discussed above, which causes peptic ulcers and cancer (Chapter 2) (Blanchard et al, 2004; Rad et al, 2004). H. pylori will often enter discussions of chronic disease. IL-10 (‘anti-inflammatory cytokine’) has a promoter polymorphism that influences secre- tion by several-fold (Yilmaz et al, 2005) and is associated with different outcomes of hepatitis, meningitis, periodontal disease, and H. pylori (Chapters 2, 4, and 5). CRP variants may interact with IL-6 variants. Four sites in the CRP gene are associated with plasma CRP levels: upstream promoter (Brull et al, 2003; Kovacs et al, 2005); exon 2 (Zee and Ridker, 2002); the intron (Szalai et al, 2002); and the 3′-untranslated region of the mRNA (3′-UTR) (Brull et al, 2003). Moreover, plasma CRP levels are influenced by alleles of IL-1 and IL-6 (Ferrari et al, 2003; Latkovskis et al, 2004) and of apoE (Section 5.7.4) (Austin et al, 2004; Rontu et al, 2006). 54 The Biology of Human Longevity
  • Inflammation and Oxidation in Aging and Chronic Diseases 55 The apolipoprotein E allele apoE4, which is infamous for increasing the risk of heart attack and dementia (Section 5.7), also influences inflammatory responses. In some contexts, apoE4 protein is proinflammatory relative to apoE3. In cultured glia, exogenous apoE4, but not apoE3, induced IL-1 secretion (Chen et al, 2005; Guo et al, 2004). After surgery, apoE4 carriers had higher blood TNFα than the apoE3 (Drabe et al, 2001; Grunenfelder et al, 2004). Transgenic models confirm these effects. IL-6, TNFα, and nitric oxide (NO) production by transgenic apoE4 mice (targeted gene replacement) was greater than with apoE3 (Colton et al, 2004; Lynch et al, 2003). In its newest function, ApoE also mediates lipid antigen presentation to T-cells through CD1 mechanisms that are independent of the MHC system (Hava, 2005; van den Elzen et al, 2005). Other lipoproteins are also important in inflammation (Section 1.4.2.2). The major histocompatibility complex (MHC) is a cluster of hundreds of genes that modulate instructive immunity and inflammation (Finch and Rose, 1995; Klein, 1986; Price et al, 1999). The MHC has many variant alleles in each of its genes and may be the most polymorphic complex locus in the human genome. The different combinations of alleles across the MHC gene complex (haplotypes) differ in proportion between human populations. MHC genes encode proteins used in antigen presentation, but also a wealth of acute phase and other inflammatory mediators including complement factors (Bf, C2, C4), HSP70, and TNFα (Finch and Rose, 1995). The MHC haplotypes are thought to represent selection for resistance to specific pathogens and toxins (Borghans et al, 2004; Klein, 1986; Wegner et al, 2004). Like IL-6, the MHC is physiologi- cally integrated. MHC allelic variants influence insulin and glucose signaling (Assa-Kunik et al, 2003; Lerner and Finch, 1991; Napolitano et al, 2002; Ramalingam et al, 1997) and reproductive cycles (Lerner et al., 1998, 1992; Lerner and Finch 1991). Because of effects on life history traits that balance trade-offs of immunity, reproduction, and metabolism across the life span, the MHC may be considered a ‘life history gene complex’ (Finch and Rose, 1995). The emerging genetics of inflammation may also involve haplotypes of the MHC. The MHC haplotypes involving TNF alleles show tentative associations with ischemic heart disease (Porto et al, 2005), whereas TNF isoform variants influence expression of the neighboring complement C4 gene (serum C4a levels) (Vatay et al, 2003). Besides the MHC gene cluster on chromosome 6 (Ch 6) with com- plement C2, C4, TNFα etc., other chromosomes include clusters of inflammatory and host defense genes: Ch 1, Regulator of Complement (RCA) cluster (C4bp, CR1 and CR2, DAF, factor H, MCP); Ch 2, interleukin-1 cluster (IL-1α, IL-1β, IL-1RN); Ch 11, apo A-I/C-III/A-IV gene cluster; Ch 16, CD11 cluster (integrins CD11a,b,c; adhesion receptors LFA-1, Mac-1, p150,95). Many other inflammatory genes for interleukins, chemokines, etc., are scattered across the chromosomes. The many inflammatory mediators with genetic variants could contribute to the multi-factorial variability of many diseases. For example, the risk of gastric ulcer in H. pylori infections is associated with particular polymor- phisms in both the CD11 cluster (Hellmig et al, 2005) and the IL-1 cluster
  • 56 The Biology of Human Longevity (Hellmig et al, 2005). The number of possible combinations among these gene variants is very large. Among the 10 or so recognized inflammatory risk factors of vascular disease, each has at least two genetic variants. Thus, the number of possible interactions approximates 2 raised to the 10th power, or 1024. This calculation gives an overestimate because some inflammatory genes are clustered on the same chromosome and hence not randomly assorted. The number of combinations grows faster for genes with more than two variants, as is the case for CRP and IL-6. These infrequent combinations could underlie sporadic cases of the major diseases that do not show obvi- ous heritability. As human genome variations become mapped in populations in greater detail, it may be possible to find inflammatory gene haplotypes of polymorphic loci on different chromosomes that were selected by infectious disease. As a precedent, combinations of alleles in eight inflammatory genes and apoE were very recently found that discriminate Alzheimer disease risk groups (Licastro et al, 2006). Variants in inflammatory genes and insulin/IGF-1 are also implicated as regulators of longevity (Franceschi et al, 2005; van Heemst et al, 2005) (Section 5.7.2). 1.3.3. Inflammation and Energy The acute phase response to infections rapidly mobilizes host energy needed to sustain fever and acute phase protein synthesis (Fig. 1.2B). White adipose tissue and liver have major roles in the energetics of host defenses (Khovidhunkit et al, 2004; Pond, 2003; Trayhurn and Wood, 2004). Plasma triglycerides increase within 2 h of infection from lipolysis in fat cells and by hepatic synthesis of fatty acids and triglycerides. Triglyceridemia may be sustained for a day or more. These generalized changes are induced by bacterial endotoxins and are medi- ated by IL-1, IL-6, and TNFα and other cytokines, which have direct metabolic effects. For example, TNFα acts directly on adipocytes to increase lipolysis and lower insulin sensitivity. IL-6 also stimulates lipolysis and in addition hepatic triglyceride synthesis. Infections cause immediate and chronic energy deficits. The energy consumed by fever comes from 25–100% increases in basal metabolism (Lochmiller and Deerenberg, 2000; Waterlow, 1984). Protein, glycogen, and fat are mobilized; e.g., human energy debts in septic infections are 5,000 kJ/d (Plank and Hill, 2003), which approximates 50% of the normal daily food intake (10,000 kJ/d or 2392 kcal/d). In patients with active infections, white blood cell oxygen consumption increased by 50%, mostly from ATP turnover (Fig. 1.2B) (Kuhnke et al, 2003). The acute phase responses induce ‘sickness behaviors’ through hypothalamic mechanisms that decrease appetite and induce lethargy. T cell activation is closely coupled to the uptake of glucose and other extra-cellular nutrients (Fox et al, 2005). While energy partitioning to various physiological and cellular processes is understood in broad outline
  • (Buttgereit and Brand, 1995; Buttgereit et al, 2000; Rolfe et al, 1999), we do not know how much of the energy consumption associated with fever is due to activation of immune cells and the increased production of CRP and other acute phase proteins. The demands of immunity are considerable, because diet restriction attenuates the primary and secondary immune responses (Chapter 3) (Martin et al, 2007). Growth in children is also attenuated by infections which impair ingestion even if food is not limited (Chapter 4). Malnourished children with infections are in energy debts of 285 kcal per day of infection (McDade, 2003). The progressively reduced load of infections and inflammation in the 19th and 20th centuries is linked to the increased growth of children (Crimmins and Finch, 2006a) (Chapters 2 and 4). White adipose tissue is considered an endocrine organ because of its many secreted peptides, particularly leptin and adiponectin (‘adipokines’), which regulate metabolism and eating behavior (Ronti et al, 2006; Trayhurn and Wood, 2004). Leptin modulates appetite by binding to neurons in hypothalamic centers that regulate energy, body temperature, and reproduction; leptin also influences on insulin sensitivity and stimulates lipid β-oxidation in skeletal muscle. Adiponectin has some overlapping activities by stimulating muscle lipid oxida- tion by skeletal, and also inhibiting hepatic gluconeogenesis. Both leptin and adiponectin activate the critical AMP-activated protein kinase (AMPK) pathway that increases glucose uptake and lipid oxidation (Chapter 3, Fig. 3.11). Leptin is highly conserved in vertebrates, with homology to IL-6 and TNFα, and binds to class I cytokine receptors (Boulay et al, 2003). Moreover, leptin directly binds CRP in human serum and blocks binding to leptin receptors (Chen et al., 2006). These highly pleiotropic activities of leptin epitomize the nexus of immunity and energy regulation. Because leptin is secreted in proportion to white fat mass, blood leptin levels are an index of energy reserves (Ronti et al, 2006; Trayhurn and Wood, 2004). Leptin is also a major immunomodulator with complex roles that are still emerg- ing (Loffreda et al, 1998; Matarese et al, 2005; Steinman et al, 2003). Endotoxin induces a leptin surge, which may be an important coordinator of the acute and chronic phases of immune responses. In macrophages, leptin stimulates phago- cytosis and increases secretion of IL-6 and TNFα. Adiponectin may have oppos- ing actions (Kougias et al, 2005). In adaptive immunity, leptin is a co-stimulant of T-cell subsets, e.g., naive CD4+ CD45RA+ T cells. Exogenous leptin can override energy decisions that attenuate immunosuppression, e.g., in starved mice infected with Streptococcus (Klebsiella pneumonia), leptin injection rapidly corrected the impaired phagocytosis (Mancuso et al, 2006). Reciprocally, diet (energy) restric- tion may be an intervention for the immunological abnormalities of obesity (Lamas et al, 2004), while fasting may benefit autoimmune disorders because of decreased leptin (Sanna et al, 2003). Another relationship of fat to immunity is found in lymph nodes, where spe- cialized adipocytes appear to supply follicular dendritic cells with fatty acids (Pond, Inflammation and Oxidation in Aging and Chronic Diseases 57
  • 2003, 2005). Perinodal adipose tissue differs from adipocytes in other locations by a greater content of polyunsaturated fatty acids and by resistance to atrophy during starvation or hibernation. Intramuscular adipocytes may also be specialized. Adipocytes also secrete acute phase proteins including CRP, IL-6, and TNFα; complement factors and serum amyloid A3 (SAA3); and prothrombotic factors plasminogen activator inhibitor-1 (PAI-1) (Lyon et al, 2003; Trayhurn and Wood, 2004). SAA-3 and PAH-1 are induced by endotoxin (LPS) and hyperglycemia (Lin et al, 2001; Lin et al, 2005). Moreover, white fat depots have numerous macrophages in proportion to obesity (body mass index) (Bruun et al, 2006; Trayhurn and Wood, 2004; Weisberg et al, 2003). The levels of inflammatory gene expression differ between white fat pad adipocytes and embedded macrophages: IL-6 is expressed in both, with TNFα more prevalent in these macrophages (Weisberg et al, 2003). The gene expression profile of white adipose tissue gives a good account for the association of obesity with the proinflammatory, pro- thrombotic blood profile in vascular events (Weisberg et al, 2003). Exercise and diet restriction can reduce the macrophage content and inflammatory expression profile of white fat (Bruun et al, 2006) (Chapter 3). The brain receives information about the inflammatory status by detecting changes in blood sugar, cortisol, and other hormones, but also directly by the vagus and other nerves going from the gut to the brain stem and hypothalamus (Besedovsky and Del Rey, 1996; Black, 2002; Tracey, 2002). These neuronal inputs are part of inflammatory reflexes, which regulate hormonal secretions by the hypothalamus and pituitary and secretion of cytokines by cells throughout the body (Black, 2002; Tracey, 2002). Inflammation activates the hypothalamic- pituitary-adrenal axis and increased blood levels of the adrenal steroid, cortisol. Cortisol is called a ‘glucocorticoid’ because it stimulates the biosynthesis of glucose (‘gluconeogenesis’) (Riad et al, 2002), using carbon fragments derived from stored protein and fat. Cortisol elevations can attenuate induction of some cytokines (Franchimont et al, 2003; Nadeau and Rivest, 2003; Refojo et al, 2003), which is part of the basis for steroidal anti-inflammatory drugs (SAIDs) such as prednisone and dexamethasone. Besides their key roles in disease, many inflammatory mediators also have normal physiological roles that are independent of the acute phase responses. During exercise, IL-6 is released by skeletal muscle in proportion to the intensity of muscular challenge (Helge et al, 2003). In adipocytes, IL-6 directly regulates insulin responsiveness (Bastard et al., 2002). Eating foods rich in fat, or induced hyperglycemia, increases IL-6 and TNFα by 50% within 4 hours (Esposito et al, 2002; Nappo et al, 2002). Could these proinflammatory responses to ingestion have been evolved as a protection against infectious pathogens that were very common in food, until recently? Chapter 6 discusses the dangers of eating raw meat in relation to our evolution. The dense synergies among the hormonal reg- ulatory systems of growth, metabolism, and immune functions may be critical to diseases that limit human life spans. 58 The Biology of Human Longevity
  • 1.3.4. Amyloids and Inflammation Amyloid proteins are closely associated with acute and chronic inflammation, and may be universally accumulated during aging.3 Amyloid fibrils are aggregates of 10 nm filaments with repetitive β-sheets in parallel to the fibril axis (Kisilevsky, 2000; Pepys, 2005; Sipe and Cohen, 2000). The cross-β structure of amyloids is detected by binding Congo red, a birefringent dye (‘congophilic amyloids’); how- ever, cross-β structures are also formed by many peptides not yet associated with amyloidosis (Carulla et al, 2005). About 20 peptides encoded by separate genes can form amyloids. Extracellular amyloid fibrils accumulate in diseases of brain (Alzheimer, Creutzfeldt-Jakob) and heart (cardiomyopathy, atheromas). During aging, nearly all tissues accumulate some amyloid (Schwartz, 1970; Tan and Pepys, 1994; Walford and Sjaarda, 1964; Wright et al, 1969). Chronic inflam- mation often induces amyloids, some of which are acute phase proteins (CRP, SAA, SAP) with anti-microbial activities, discussed below. Bystander effects are also shown, with the accumulation of AGEs and other oxidative damage that attract macrophages and activate scavenger receptors (Section 1.4.4). Three amyloids are notoriously associated with tissue damage: Aβ (amyloid β-peptide) of Alzheimer disease, amylin of diabetes, and transthyretin in cardiomyopathy. Amyloid deposits are usually embedded with acute phase pro- teins and sulfated proteoglycans (heparin). The amyloid bulk disrupts functions in the hereditary transthyretin amyloidoses, which damage the myocardium and peripheral nerves (Buxbaum and Tagoe, 2000; Morner et al, 2005). Oligomeric amyloid aggregates are also cytotoxic (Bucciantini et al, 2002; Kayed et al, 2003; Reixach et al, 2004) and important in Alzheimer disease (Section 1.6). Other amyloidogenic proteins are acute phase responses and some have anti- microbial activities. C-reactive protein (CRP), serum amyloid A (SAA), and serum amyloid P (SAP) bind microbial pathogens. These ancient proteins form pentameric fibrils (pentraxins) that have orthologues throughout vertebrates and invertebrates (Finch and Marchalonis, 1996; Shrive et al, 1999; Ying et al, 1992). The case for anti-microbial functions is clearest for CRP, which enhances the phagocytosis (opsonization) by binding lipopolysaccharide (LPS) and other com- ponents of gram-negative bacteria (Bodman-Smith et al, 2002; Ng et al, 2004). Infections can induce amyloids throughout the body, including the brain, as observed in HIV (Section 2.7.2). In a transgenic model of transthyretin, myocar- dial amyloid was observed in one mouse colony considered ‘dirty,’ but not in a specific-pathogen free (SPF) colony (Noguchi et al, 2002). Moreover, tissue Inflammation and Oxidation in Aging and Chronic Diseases 59 3 Rudolf Virchow, founder of the cell theory, introduced ‘amyloid’ in 1854 to describe starchy hepatic deposits that reacted with iodine. However, this histochemistry was mis- leading: amyloids were soon identified as proteins (Andree and Sedivy, 2005; Picken, 2001), but the old term persists.
  • amyloids in DBA/2 mice were observed in earlier dirty colonies, but not in the cleaner later SPF colonies (Lipman et al., 1993). These responses to the micro- bial environment suggest that tissue amyloid deposits may be part of host defense innate immunity. On the other side, some infectious bacteria and fungi have evolved cell-wall amyloid proteins to penetrate host defenses (Gebbink et al, 2005). The microcin amyloid of Klebsiella pneumonia forms cytotoxic pores, as well as non-toxic fibrils (Bieler et al, 2005), whereas the Tafi peptide of Salmonella typhimurium forms amyloid fibrils that enhance intestinal colonization (Sukupolvi et al, 1997). Endless host–pathogen “arms races” that select for specialized host responses may give rise to population-specific genetic risk factors in Alzheimer and other amyloidotic diseases. 1.4. BYSTANDER DAMAGE AND DEPENDENT VARIABLES IN SENESCENCE Bystander damage is fundamental to aging. Inevitably, long-lived molecules accumulate damage from chemical agents in the immediate fluid environment, particularly glucose and free radicals such as reactive oxygen species (ROS) (Fig. 1.11). This chemical damage is described as ‘bystander’ because it is pas- sively incurred. Bystander damage accumulates when the rate of incident damage exceeds the rate of molecular repair by enzymes or by ‘rejuvenation’ from new molecular synthesis with catabolism (turnover) of the old molecule. Bystander damage with time-dose relationships is recognized by epidemiology, e.g., pack-years of exposure to tobacco smoke (Chapter 2). Other biomedical fields have equivalents. Arterial disease includes bystander damage, as an ‘aging process × exposure time interaction’ (Lakatta and Levy, 2003a). Different types of bystander effects arise during immune clone differentiation through diffusible immunoregulators (Fletcher et al, 2005). It seems fruitful to expand bystander damage as a framework for resolving the levels of environmental effects in aging, exogenous and endogenous. Four types of bystander damage may be considered. Type 1: Free radical damage, which is intensified by inflammation (Chapter 2) or attenuated by diet restriction (Chapter 3). Inflammation can also induce tissue amyloid deposits, which then become bystander targets. Type 2: Glyco-oxidation of proteins and DNA occurs non-enzymatically by spontaneous reaction with sugars that are omnipresent in extracellular fluids (AGE, or advanced glycation endproducts). Glyco-oxidation also stimulates local production of reactive oxygen species (ROS) and other inflam- matory responses in arterial remodeling. Type 3: Chronic cell proliferation, which can be stimulated by oxidative stress and inflammation, leading to increased somatic mutational load, increased telomere erosion, and altered immune functions (depletion of naive T cells). Type 4: Mechanical trauma, which accumulates 60 The Biology of Human Longevity
  • unavoidably during aging in the real world through accidents and mechanical wear and tear, but also violence from predation and social stress. At the micro- scopic level, mechanical forces in arterial blood flow (atheroprone flow) influence the location of atheromas through inflammatory patterns of gene expression (dis- cussed briefly below and extensively in Section 1.5.3.2). This outline of bystander damage as a main category of molecular aging is an initial draft of concepts that cannot be comprehensive. Bystander damage may be understood in distinction with two other types of molecular aging changes that are less dependent on the immediate environment: The spontaneous racemization of L-amino acids and the misincorporation (tem- plate errors) in DNA synthesis both occur at some irreducible rate that may be considered as ‘intrinsic aging’. Amino acid racemization is an intrinsic property of covalent bond instability, which would also occur in a vacuum. The accumu- lation of D-aspartate may be used to estimate the age of the protein, with some caveats (Bada et al, 1974; Helfman and Bada, 1975). Mutations and other errors in DNA replication, transcription, and translation are inevitable because enzymes have intrinsic and irreducible errors in templating. In DNA polymerases, chain elongation depends on selection of the properly matched base among the four choices (AT,GC), which arrive at the site of synthesis by diffusion. Selection of the next base for chain elongation depends on Watson-Crick complementary pairing, which is entropy-driven (Petruska and Goodman, 1995). The role of entropy to telomere DNA erosion during somatic cell replication is undefined. Errors in transcription and translation may involve similar issues in mismatching, but much less is known (Finch and Kirkwood, 2000). Nonetheless, racemization and misincorporation are subject to the local environment. D-amino acids can be removed by repair enzymes (Brunauer and Clarke, 1986; DeVry et al, 1996). While DNA repair is well studied (base excision repair of oxidized bases), oxidized amino acids in proteins are also repaired. Repair mechanisms and chem- ical defenses against bystander damage may be fundamental in the evolution of life spans. 1.4.1. Free Radical Bystander Damage (Type 1) Free radicals cause chemical damage with long-term consequences of increased mortality, as shown in examples discussed in the following chapters. Lungs are vulnerable to bystander damage with further progressive consequences to heart functions. Cigarette smoke causes chronic oxidative stress from activated macrophages (MacNee, 2001). Particulate aerosols from the combustion of fossil fuels (e.g., ‘oil fly ash’) cause oxidative damage and chronic respiratory disease (Ghio et al., 2000a,b, 2002). Fly ash inhalation stimulates invasion by leukocytes, which release superoxide; lung fluids had 2-fold higher levels of TNFα (which activates neutrophils) and higher GSSG (oxidized glutathione, reflecting oxida- tive stress). Lung damage is greatly attenuated by increased extra-cellular SOD in transgenic mice (Ghio et al, 2002). Increased SOD also blunts lung damage to Inflammation and Oxidation in Aging and Chronic Diseases 61
  • hypoxia (Ahmed et al, 2003). Moreover, nitric oxide deficits (NOSII gene deletion) (Fakhrzadeh et al, 2002) or deficits of TNF-receptors (Cho et al, 2001) decrease lung inflammatory damage after ozone inhalation. Systemic oxidative damage is caused by exposure to other inflammogens and infections. Rodents injected with the endotoxin LPS to cause sterile inflammation had rapid 5-fold increases of plasma LDL hydroperoxides (Memon et al, 2000). Lipoprotein oxidation during infections has important impact on atherogenesis, discussed below. Because oxidatively damaged molecules are recognized by macrophages through scavenger receptors, the induction of inflammatory genes during aging (Section 1.8) could be downstream to these host defenses. The uptake of oxidized LDL by vascular macrophages is a ‘molecular Trojan horse’ that induces inflammatory processes at the core of atherogenesis (Hajjar and Haberland, 1997) (Section 1.6). Thus, many aspects of aging could result from cell and molecular damage from extra-cellular free radicals produced during chronic, low-grade inflammation (Query II). Chronic infections also cause chronic inflammatory bystander damage through ROS and other free radicals. Among many examples, infections by the enter- obacter Helicobacter pylori cause local inflammatory responses that increase DNA oxidation, cell proliferation, and the mutational load, and are a major cause of gut cancer (Section 2.8.1). Another classic example is tuberculosis (TB), which causes organ damage by host defense mechanisms that induce local fibrous con- nective tissue to wall off the bacillus and local amyloid deposits (Nathan et al, 2002). Pulmonary TB typically causes loss of lung tissue and ‘vital capacity,’ which is a major risk factor of mortality during aging (Section 1.2.2). TB also increases systemic oxidation, with higher lipid peroxidation in serum proteins and erythrocytes (Vijayamalini and Manoharan, 2004). Amyloids (Section 1.3.4) are a crucial interface of inflammation and oxidative bystander damage through local oxidative stress (Ando et al, 1997; Butterfield et al, 2002; Miyata et al, 2000; Wong et al, 2001). The AA-amyloid fibrils in tuber- culosis (TB) and rheumatoid arthritis (RA) and in a mouse model of SAA accu- mulate advanced glycation endproducts (AGEs): carboxymethyllysine (CML), and 4-hydroxynonenal (4-HNE, lipid peroxidation adduct) (Kamalvand and Ali-Khan, 2004). In Alzheimer brains (Girones et al, 2004; Reddy et al, 2002; Wong et al, 2001) and in a transgenic model (Munch et al, 2003), fibrilar Aβ amyloid also accumulates glyco-oxidation and lipid peroxidation adducts. In the glia sur- rounding brain Aβ deposits (Fig. 1.10B), AGEs are colocalized with iNOS (Wong et al, 2001) and may activate microglia through RAGE receptors (Section 1.2.6). Metals may have a particular role in senile plaque amyloid, which has copper, iron, and zinc at concentrations 2- to 5-fold above healthy brain neuropil (Lovell et al, 1998). Aß and metal interactions are implicated in oxidative damage (Bush and Tanzi, 2002). The Aß peptide directly binds iron, which increases cytotoxic H2 O2 production (Boyd-Kimball, 2004). Moreover, Aß binds heme to form com- plexes with peroxidase activity (Atamna and Tanzi, 2006). Furthermore, trace metals promote Aß aggregation (Huang et al, 2004c). There is little doubt that accumulated 62 The Biology of Human Longevity
  • adducts in amyloids are proinflammatory, but their contribution to cell death is not defined. Age-related increases of mitochondrial production of ROS through proton leak in rat liver and muscle causes bystander damage (Section 1.2.6). Diet restriction attenuates mitochondrial ROS production and DNA damage (Chapter 3). These benefits of diet restriction may be due to lower insulin and glucose, because mito- chondrial ROS production is sensitive to insulin (Lambert et al, 2004). Conversely, maturity onset diabetes is associated with increased mitochondrial DNA damage and impaired energetics (Wallace, 2005). 1.4.2. Glyco-oxidation (Type 2) Oxidative damage to DNA, lipids, and proteins is the result of unavoidable expo- sure to blood glucose that chemically generates AGEs (Section 1.2.6). The pro- duction of AGEs is increased by hyperglycemia and decreased by diet restriction (Chapter 3). Moreover, cooked food is an important source of AGEs, which can induce systemic inflammatory responses (Section 2.4.2). These experiments imply a large role for inflammation-induced ROS damage, which has been neglected in discussions of oxidative damage during aging because of the major focus on mito- chondrial ROS. Because AGEs inevitably accumulate from the constant exposure of long-lived molecules to omnipresent glucose, bystander damage seems appli- cable to early stages of these general aging processes. The conceptualization of AGE formation as bystander damage is my own and open to discussion. 1.4.3. Chronic Proliferation (Type 3) Adaptive immunity changes profoundly during aging as memory T cells increase at the expense of naive T cells. The depletion of naive T cells by chronic infections (Section 2.8) could be included in bystander effects. As noted in the overview of immune aging (Section 1.2.2), chronic infections by the ubiquitous virus CMV are associated with the ‘immune risk phenotype’ of impairments in the elderly, which increases mortality risk (Akbar and Fletcher, 2005; Pawelec et al, 2005). Unexpectedly, these studies also associated CMV seropositivity with greater differentiation of CD4+ T cells specific for other anti- gens than in CMV-seronegative elderly (Fletcher et al, 2005). Other CD4+ T cell specificities included varicella zoster virus (VZV) and Epstein-Barr virus (EBV). The greater T cell differentiation was characterized by shorter telomeres and loss of CD27 and CD28 costimulatory proteins. It was hypothesized that IFN-α and TNFα are secreted during activation of CMV-specific T cells and diffuse to accelerate other T-cell differentiation ‘in a bystander fashion.’ This concept was validated with CMV-activated T cells and T cells of other specificities, in which the inhibition of telomerase was shown to depend on IFN-α. Further evidence is the acceleration of T-cell differentiation by IFN-α therapy for hepatitis-C virus, which increased the proportion of CD28− T cells (Manfras et al, 2004). Inflammation and Oxidation in Aging and Chronic Diseases 63
  • In a different human population, (Khan et al, 2004) showed that CMV infection reduced immunity to EBV. Thus, chronic immune activation by CMV and pos- sibly other common antigens can cause bystander effects by accelerating the differentiation of other T cells through secretion of IFN-α and other cytokines. The increased secretion of proinflammatory cytokines and the extreme T-cell differentiation during CMV infections may be an important link to the ‘immune risk phenotype.’ Telomere erosion can be affected by bystander damage through oxidative stress. For example, telomere shortening is accelerated by oxidative stress in vas- cular endothelial cells (HUVECS) (Kurz et al, 2004). In diploid fibroblasts, telom- ere loss was attenuated by overexpressing extracellular SOD (EC isoform), which also lowered intracellular peroxides that can cause oxidative damage; the increased SOD also increased the cell replicative potential (Serra et al, 2003). SOD may decrease single-strand DNA breaks induced by oxidative stress, which enhances replicative telomere DNA loss (Sitte et al, 1998). Telomere erosion is pertinent to arterial disease because the replicative senescence of endothelial progenitor cells may be accelerated by elevated antiotensin in hypertension, or attenuated by antioxidants and statins (Section 1.5.3). Yet other conditions shorten telomeres without primary causes of single-strand DNA breaks or other DNA damage by oxidative stress. Local secretions of cytokines during differentiation of T cells may cause other bystander effects by influencing the differentiation of neighboring immune cells (Fletcher et al, 2005) (Section 2.5.1). Inhibition of the glutathione-dependent antioxidant system that disposes of peroxides also accelerated telomere shortening and shortened the replicative potential in endothelial cultures (Kurz et al, 2004). Telomere length may represent cumulative stress as bystander exposure (von Zglinicki and Martin-Ruiz, 2005). In different tissues of the same elderly individ- uals, telomere length correlated strongly in fibroblasts and blood monocytes (Friedrich et al, 2000; von Zglinicki et al, 2000). The absolute lengths of telom- eric DNA differed widely, but individuals with long telomeres in monocytes had long telomeres in their fibroblasts. The cell correlations in different tissues within an individual imply systemic influences on cell proliferation, with consequent impact on telomere DNA in multiple cell types. The cumulative exposure to infections (Section 2.5.1) and stress during the lifetime (Section 2.5.2) may deter- mine the overall levels of telomere erosion in lymphocyte clones. Systemic inflammatory responses can influence metabolic hormones, some of which regulate telomerase activity—e.g., IGF-1 (Bayne and Liu, 2005). 1.4.4. Mechanical Bystander Effects (Type 4) Blood pulses transmit mechanical forces to the arterial endothelium that causes local molecular and cell responses as bystanders in this most vital function. Atheromas tend to form at arterial branches and curves, where the physics of blood flow alters shear forces. Growing plaques are increasingly exposed stresses 64 The Biology of Human Longevity
  • that may cause fissures and fractures. Thus, the direct impact of blood flow may cause plaque instability through mechanical forces as bystander effects. Moreover, arterial flow variations ‘atheroprone’ and ‘athero-protective’ induce inflammatory gene expression in the vascular endothelia (Dai et al, 2004)—e.g., IL-1 and com- plement C3—but decreased expression of IL-10, an anti-inflammatory cytokine. The redox-sensitive transcription factor NF-κB is 4-fold higher in atheroprone regions. However, normal flow represses arterial inflammatory gene expression. These examples of bystander damage suggest a casual framework for con- sidering aging as a system of interactions, exogenous and endogenous, rather than autogenous. Recognizing that bystander damage is a major outcome of aging leads to a broader issue, about time as an independent variable in ‘age- related’ changes of senescence. Aging and time in this sense are operationally the ‘duration of exposure.’ In essence, most aging changes are event-dependent, rather than time-dependent. Event-dependence helps focus on the proximal causes of changes during ‘aging’ (Finch, 1988; Finch, 1990, p. 6). This recogni- tion is explicit in epidemiological models, for example, of the cancer risks of smoking, which consider pack-years, rather than age-years of smoking. Dose- duration relationships are also understood as fundamental in arterial disease (area of artery involved, Fig. 1.6A); in colo-rectal cancer (area of intestine inflamed, Section 2.9.1); and growth attenuation by enteric infections (diarrhea days, Chapter 4.6.1). Each system of bystander damage may have boundary values in the commutative product of dose × duration, because of threshold effects and excluded values. PART II The second part of Chapter 1 considers in more detail the workings of inflam- matory processes at work in arterial disease and Alzheimer disease, and their over- lap with aging change in the absence of diseases. Shared inflammatory processes may mediate effects of diet, drugs, and lifestyle (Chapters 2 and 3); may have been the basis for the recent increases of life span in human populations (Chapter 2); may be in the developmental origins of arterial disease and diabetes (Chapter 4); may be influenced by genetic variants in aging and life span; and may have been the basis for the evolution of the longer human life span from great ape ancestors (Chapter 6). The slow degenerative changes involve complex remodeling processes in arteries and brain that extend beyond simple oxidative damage. 1.5. ARTERIAL AGING AND ATHEROSCLEROSIS The primacy of arterial degeneration in human aging and the importance of inflam- matory processes are not modern concepts. A century ago, William Osler asserted the importance of arterial degeneration in human aging: “Arterio-sclerosis is an Inflammation and Oxidation in Aging and Chronic Diseases 65
  • accompaniment of old age, and is the expression of the natural wear and tear to which the tubes are subjected. Longevity is a vascular question, which has been well expressed in the axiom ‘a man is only as old as his arteries’” (Osler, 1892, p. 664). Even in 1858, Rudolph Virchow considered inflammation as a primary cause of arterial disease: “I have . . . no hesitation in siding with the old view . . . in admitting an inflammation of the arterial coat to be the starting point of . . . athero- matous degeneration. . . . we have here an active process which really produces new tissues, but then hurries on to destruction in consequence of its own devel- opment,” translated and cited by (Langheinrich and Bohle, 2005). After one million more scientific reports on vascular disease, these early insights are well validated: Arterial aging is fundamentally an inflammatory process from its beginnings before birth over the life span (Ross, 1995, 1999). 1.5.1. Overview and Ontogeny The main seats of vascular aging and atherogenesis are in the arterial endothelia and the elastic lamina, which interact with multifarious inflammatory influences from the internal and external environment (Fig. 1.2A). The aorta and other cen- tral arteries are elastic reservoirs for the blood volume expelled at each heart- beat; the elasticity declines progressively during aging. The subsidiary arterioles have relatively more smooth muscle, which modulates blood pressure by adjusting the diameter (lumen), contracting in response to adrenaline, or relax- ing in response to nitric oxide, among other signals. Two distinct, but interre- lated, processes operate upon arteries throughout life4 (D’Armiento et al, 2001; Najjar et al, 2005; Wang et al, 2006). (I) Arterial aging is a generalized thicken- ing and stiffening of arterial walls that slowly increase blood pressure and may lead to hypertension. (II) Atherogenesis is a local (patchy) growth of cells and accumulation of lipids within the arterial wall consisting of a progression from microscopic foci, to fatty streaks, to raised plaques that become fibrous and cal- cified.5 Vascular pathologists refer to ‘plaques’ in reference to developed vascu- lar lesions, described in eight types or grades (Fig. 1.13). Atheromas arise focally as ‘responses to injury,’ in Ross’s concept (1999). Large atheromas may cause stenosis by intruding into the lumen and decreasing blood flow. Clots can form on the atheroma surface, or circulating clots can be trapped, leading to 66 The Biology of Human Longevity 4 My main sources of information and insights on vascular aging are Edward Lakatta, Claudio Napoli, and Wulf Palinski, discussions and papers. 5 Atherosclerosis refers to pathological thickening of the inner arterial wall, while the older term arteriosclerosis (‘hardening of the arteries’) includes generalized arterial aging changes (loss of elasticity and intima-media wall thickening, Table 1.4) that may not be immediately associated with pathology. Arteriolosclerosis refers to aging changes in smaller arteries. Arterial plaques should not be confused with brain senile plaques in Alzheimer disease, although both arterial and Alzheimer plaques share many inflammatory processes (Table 1.3).
  • I. Microscopic foci; foam cells and oxLDL Fetal aorta and later; may regress, or not progress May cause occlusion Lipid regression in IV-V may lead directly to VII-VIII II. Multiple layers of foam cells III. Isolated extracellular lipid pools IV. Confluent lipid pools; early calcium V. Fibromuscular layers Relativeriskofvascularevent VI. Erosion and instability; thrombosis VII. Extensive calcification VIII. Extensive fibrous changes Coronary artery at lesion-prone location Type III (preatheroma) Small pools of extracellular lipid Adaptive thickening (smooth muscle) Fibrous thickening Thrombus Fissure and hematoma Intima Media Type VI (complicated lesion)Type V (fibroatheroma) FIGURE 1.13 Natural history of atheromas from fatty streak to advanced lesions, rupture, calcifi- cation, and regression. American Heart Association grades, adapted from (Stary, 2000) and (Strauss et al, 2004): A simplified schematic of atherogenesis, beginning as microscopic foci in the embryo and growing progressively during childhood into adult life. This diagram depicts cycles of endothelial injury, lipid deposits, macrophage influx, and quiescence. I, isolated macrophage foam cells; II, mul- tiple foam layers, but arterial structure not disrupted; III to IV, addition of expanding extracellular lipid pools and cholesterol crystals (‘atheromas’); V, addition of fibromuscular layers (‘fibroatheromas’); VI, fissured and thrombotic fibrous plaques; VII, calcification; VIII, fibrotic lesions without lipid cores. Advanced plaques may rupture, attracting platelets and forming a thrombus. Ruptured plaques may be clinically silent and then heal, some remaining innocuous for years.
  • thromboses, which block arterial blood flow, in turn, causing tissue oxygen deficits (ischemia) and tissue damage (necrosis). The arterial system has a complex biochemistry, cell biology, and physiology evolved to serve tissue needs across a huge range of challenges in the demand for blood nutrients and oxygen, and the urgencies of hemostasis during pene- trating injury. Arterial walls consist of concentric layers of cells and extracellular matrix (‘connective tissue’). The innermost layer, the intima, consists of endothe- lial cells exposed directly to the blood, which are covered by a glycocalyx, as thin mesh of glycoproteins and proteoglycans (HSPGs). The endothelial lumen surface contains mechanotransducers that are sensitive to sheer stress and regulate inflammatory genes in the endothelia (Section 1.5.3.2). The other endothelial face attaches to the elastic lamina, which is also a phys- ical barrier to the pressure-driven diffusion of blood lipids and proteins. Next inside is the media, the thickest layer, with smooth muscle cells and matrix. The outermost layer is the adventitia, a sheath of matrix proteins, nerves, and capil- laries. The extracellular matrix consists of elastin (its main component), collagen, fibronectin, and mucopolysaccharides; these extracellular materials account for 50% of mass in large arteries and are targets of modification during aging. During development, arterial collagen is enzymatically cross-linked by lysyl oxidase. After puberty, further cross-links are chemically added. Arterial wall thickness is measured as the intima-media thickness (IMT) by ultrasonography. Carotid artery IMT increases universally during aging, by 2–3-fold across the life span in community studies (Fig. 1.14A) (Najjar and Lakatta, 2006; O’Leary et al, 1999). The arterial wall thickening and stiffening during aging are not benign over long life spans and predict individual risk of heart attacks, stroke, and hypertension. The top quintile of carotid IMT has 3-fold higher risk of myocar- dial infarcts (Cardiovascular Health Study) (O’Leary et al, 1999) (Fig. 1.14B). Combined, the wall thickening, elastin fragmentation, and cross-linking are the main causes of the universal increases of systolic pressure during aging. Low carotid elasticity (top quartile) was associated with 3-fold higher risk of future hypertension (ARIC Study) (Liao et al, 1999). Arterial aging rates strongly indicate future vascular health (Safar, 2005; Sagie et al, 1993). Arterial wall thickening is due to active remodeling from endothelial cell growth and fibrosis with matrix deposition of collagen and proteoglycans (Fornieri et al, 1992; Sims et al, 2001; Sims et al, 2002; Wang et al, 2003) (Fig. 1.15A). Matrix metaloproteases (extra-cellular endoproteases, e.g., MMP-2 and -9) increase several-fold across adult ages in humans, primates, and rodents (D’Armiento et al, 2001; Lakatta and Levy, 2003a; Wang et al, 2003a). In the human thoracic aorta, collagen increases by 50% from 20 to 80 y, whereas elastin declines by 35% (Faber and Moller-House, 1952). Of major importance, elastin fibers become irreversibly fragmented with aging. The aging rat aorta intimal layer also thickens 5-fold, with increased matrix (Li et al, 1999). These processes are independent of atheromas. Wall thickening during aging is comparable in human populations with relatively little atherosclerosis and in arteries 68 The Biology of Human Longevity
  • of aging rodents that do not develop atheromas during aging without major lipid perturbation (Najjar et al, 2005). However, generalized arterial aging changes may predispose to atherogenesis. Arterial rigidity and loss of elasticity may be attributed to several distinct processes consistent with bystander damage I and II. The fragmentation of elas- tic fibers may be the largest factor in loss of elasticity. Elastin is a very long-lived Inflammation and Oxidation in Aging and Chronic Diseases 69 1.0 0.0 0.2 0.4 0.6 0.8 60 70 80504030 ACCAIMT(mm) Y No CAD Possible CAD-1 Possible CAD-2 Definite CAD CTL DR BLSA CRON 0 B 1 2 3 4 5 6 Y 70 75 80 85 90 95 100 CumulativeEvent-freeRate(%) 5th 4th Quintile 3rd 2nd 1st FIGURE 1.14 Carotid artery thickening during aging; risk of myocardial infarction and stroke. A. Thickness of the common carotid artery (CCA) wall increases progressively with age (IMT, common carotid intima-media thickness). BLSA (o, Baltimore Longitudinal Study of Aging; 507 Ss, ages 42.7–85.3) (Nagai et al, 1998). BLSA subjects are mostly from upper socioeconomic strata; cardio- vascular status is assessed by electrocardiographic (ECG) response to maximum treadmill exercise; all Ss reached >85% heart rate maxima for healthy age norm; none had stenosis >50% or local plaque near carotid bifurcation. No CAD (coronary artery disease) was the largest subgroup (80%). CAD-2 had silent myocardial ischemia, with IMT overlapping Definite CAD. These benchmark data suggest benefits from diet restriction (DR) in humans: CRON study (squares, Caloric Restriction Society; 18 Ss, mean age 50) who have maintained DR >3 y; controls (CTL), conventional diet matched for age and SES (Fontana et al, 2004) (Section 3.2.3). Each 0.1-mm IMT increases CAD risk by 1.91 (Fig. 1.6C). The DR CRON study IMT are slightly above the No CAD-BLSA. B. Carotid thick- ening as a risk indicator of myocardial infarction and stroke by quintiles of intimal plus medial layers thickness. Note the 5-fold difference in risk between the highest (25%) and lowest quintiles (5%) of IMT thickness. (Adapted from O’Leary et al, 1999.)
  • protein (Fig. 1.6D). Mature elastin fibrils may not ever be regenerated in adult central arteries. Elastin fragmentation is associated with foci of increased extra- cellular protease activities: MMP-2 activity was higher near breaks in the elastic lamina of aging rat aortas (Li et al, 1999). As noted above, MMP-2 generally increases during arterial aging. Elastin fragments are thought to activate elastin- laminin receptors on endothelial and smooth muscle cells, leading to the observed increased secretion of proteolytic enzymes and enhanced local cell proliferation (Duca et al, 2004; Najjar et al, 2005; Robert, 1996). A plausible sequence is that 70 The Biology of Human Longevity 0 50 PositiveSelections(%) 100 Fetuses Children Adults Elderly MCAMCA CC AA A MDA2 HAM56 MMP CC AA MCA CC AA MCA CC AA 0 100 600 500 400 300 200 0 10 30 807060504020 B Y 0 100 600 500 400 300 200 0 10 30 807060504020 Y r2 =0.984 r2 =0.972 Common Carotid ArteryAbdominal Aorta 0 20 100 80 60 40 0 10 30 807060504020 Y Middle Cerebral Artery Exponential r2 =0.984 Cumulativelesionarea(103mm2) FIGURE 1.15 Atherogenesis in the large arteries across the life span. Redrawn from (D’Armiento, 2001): abdominal aorta (AA), middle cerebral artery (MCA), common carotid artery (CCA). A. Serial arterial sections were immunostained for macrophages (HAM-56), oxidized LDL (malondi- aldehyde, MDA2), and matrix metaloproteinase-9 (MMP). B. Lipid-rich area on arterial surfaces increase progressively with age, with suggestions of exponential increase in the middle cerebral artery and basilar artery (not shown here).
  • Inflammation and Oxidation in Aging and Chronic Diseases 71 elevated TNFα induces MMP-2, which degrades elastin. Elastin receptor activa- tion also inhibits synthesis of nitric oxide (NO), a key vascular regulator, as noted above. Elastin fragmentation may also be promoted by hemodynamic forces (O’Rourke hypothesis) (Section 1.5.3.2) Aging arteries have increased infiltration by blood cells, lipids, and pro- teins. Human coronary arteries show increased staining for gamma globulin in the intimal layer, with infiltrating lymphocytes, especially around athero- mas (Sims et al, 2001). In rat aorta, the permeability coefficient for albumin nearly doubled, 10 to 30 m (Belmin et al, 1993). Inflammatory gene expres- sion increases by endothelial and smooth muscle cells during aging; e.g., cytokines in 2-year-old rats were ≥3-fold higher IL-1, -6, -17, and TNFα (Csiszar et al, 2003). Cell death markers increase, e.g., 5-fold more apoptosis in endothelial cells together with increased caspases −3 and −9 (Csiszar et al, 2004; Ungvari et al, 2004). Aging increases MMP-2 induction in response to IL-1α and TNFα (Li et al, 1999). The chemotactic protein MCP-1 (monocyte chemoattractant protein-1) and its receptor CCR2 increased in aging rat aorta smooth muscle cells (Spinetti et al, 2004). CCR2 gene variants influenced risk of myocardial infarction in association with serum MCP-1 levels (Framingham Heart Study) (McDermott et al, 2005). An MCP-1 promoter variant was asso- ciated with 5-fold greater susceptibility to tuberculosis (Flores-Villanueva et al, 2005) and may influence the impact of infections on vascular disease (Chapter 2). Other gene sets indicate a regular sequence of changes in gene expression during arterial aging (Karra et al, 2005). Decreased availability of the free radical nitric oxide (NO) is important to declining arterial function. Endothelial NO is a key vasodilator and inhibits local platelet aggregation. Moreover, NO inhibits endothelial cell apoptosis in response to oxidized LDL and hyperglycemia. The age-related decrease in NO bioavail- ability is progressive (McCarty, 2004; Ungvari et al, 2004). The superoxide anion, with opposing actions as a vasoconstrictor, increases in aging rat arteries (Csiszar et al, 2002; Hamilton et al, 2001), which reduces NO bioavailability (Fig. 1.11) and may account for increased oxidative stress (3-nitrotyrosine) in arterial walls during aging. NO deficits are accelerated by hypertension (Hamilton et al, 2001; Taddei et al, 2001). Atheromas arise as progressive focal accumulations of inflammatory cells, lipids, and other debris. Atherogenesis and vascular occlusion occur in several stages that progress from fatty streaks to fibrous plaques to more complex raised lesions with calcification (Fig. 1.13) (Strong et al, 1999). Other pathways to rigidity involve cross-linking and calcification. Collagen and other matrix materials secreted by endothelia become cross-linked by non-enzymatic gly- cation (AGE) (Section 1.2.6). Calcification may become universal after midlife (Blumenthal et al, 1944), in association with the mineralization of elastic fibers (‘elastocalcinosis’) and increased pulse wave velocity (Dao et al, 2005). Overall wall thickening by cell growth and matrix deposits should also increase rigidity.
  • 72 The Biology of Human Longevity Atheromas may be associated with gaps in the inner (subendothelial) elastic lamina, where there is observed infiltration of macrophages, lipids, and other blood substances. Gaps of the inner lamina increase with age, particularly in human coronary arteries (Sims et al, 2001; Sims et al, 2002). Patchy loss of endothelial cells also occurs in coronary arteries (Davies et al, 1988; Sims et al, 2002). As atheromatous plaques advance, LDL cholesterol accumulates in charac- teristic ‘foam cells,’ which derive from vascular smooth muscle cells and from invading macrophages. Extracellular proteases facilitate cell migrations from the blood into atheromas and of medial cells into the intima (Garcia-Touchard et al, 2005). Cell adhesion molecules (CAMs) on the endothelia increase binding of macrophages and platelets. Advanced plaques have large deposits of lipids, cholesterol crystals, and necrotic debris in the atheroma core (Fig. 1.13). Atheromas are not randomly located, but are most frequent at arterial branches and along inner curves, where the vascular geometry disturbs lami- nar and slows blood flow (Blumenthal et al, 1954). The localization of athero- mas by the physics of blood flow to zones of low shear stress has been elegantly developed (Dai et al, 2004; Moore et al, 1994; Traub and Berk, 1998; Wootton and Ku, 1999). A striking example is the carotid arteries, where atheromas arise only in small zone within the carotid sinus. Levels of sheer stress regulate patterns of gene expression in vascular cells that are ‘athero- prone’ and proinflammatory, as described below. These localized inflammatory responses are considered as ‘responses to injury’ (Ross, 1995, 1999), consistent with bystander process that are both physical (sheer stress) and biochemical (oxidant stress, inflammation). Slow plaque growth gradually narrows the lumen of medium to large arteries (Fig. 1.13). Plaques with fibrous caps and smooth muscle cell proliferation are more stable and less prone to thrombosis (Fuster et al, 2005; Moore et al, 1994; Ross, 1995; Virmani et al, 2003; Wierzbicki et al, 2003). Plaque growth induces remodeling changes in the arterial wall, which is considered adaptive. In response to local blood flow changes, the arterial volume expands locally to alleviate local constriction. New collateral vessels are often formed (vasa vasorum) (Epstein et al, 2004). These compensations may serve remarkably well for the progressive stenosis, up to some critical point. However, plaques may become unstable and rupture, attracting platelets and triggering clotting or atherothrombosis (Fig. 1.13) (Fuster et al, 2005). Blood flow shear stress caused by protrusion of the plaque into the lumen may precipitate plaque instability on the plaque wall (bystander damage type 4, Section 1.4.4) (Slager et al, 2005). Unstable (vulnerable) plaques have thin caps and necrotic cores with macrophages and lipid deposits. The higher levels of apoptosis (induction of caspase-3) in vascular endothelia may enhance thrombus formation (Durand et al, 2004). Caps may be weakened by matrix metaloproteinases and other extracellular proteases that degrade collagen, elastin, and fibronectin and that are secreted by endothelia and by infiltrating macrophages, mast cells, and neutrophils (Lindstedt and Kovanen, 2004). Increased MMP-9 is associated with
  • Inflammation and Oxidation in Aging and Chronic Diseases 73 plaque instability (Loftus et al, 2000). Adaptive immunity with B- and T-cells may have a major role in plaque degeneration (Section 1.5.3.1). Clots (thromboses) tend to form on uneven surfaces of atheromas, which may not be symptomatic (‘silent ischemia’) until there are demands for increased blood flow. Thrombus formation may not immediately cause critical ischemia and can induce further local changes (Henriques de Gouveia et al, 2002). Far worse, clots may release fragments into the circulation that can completely block blood flow in smaller arteries and arterioles (thromboses), causing heart attacks or strokes (Libby, 2003). About 50% of infarcts are attributed to clots from small atheromas. Elevated blood fibrinogen, another inflammatory response, favors clotting. Plasma CRP elevations may be markers of unstable plaques (Schwartz et al, 2003). Ultrasonography is approaching cellular levels of resolution to detect plaque stability (Fuster et al, 2005; Langheinrich and Bonle, 2005; Tuzcu et al, 2001). Aneurysms are another inflammation-related lesion. These outpocketings of the outer wall are filled with blood under pressure and may burst with fatal effects. The abdominal aorta is the most common locus, affecting 5% or more by age 65 (Palinski, 2004; Wanhainen et al, 2005). Aneurysms involve the medial layer, with smooth muscle cell atrophy and inflammatory changes extending to the outer adventitial layer (Palinski, 2004; Zhao et al, 2004). Adventitial changes include increased 5-lipooxygenase (macrophages, mast cells), which produces proinflammatory leukotrienes from arachidonic acid; and MMPs, which may weaken the medial layer. Little is known about mechanisms that may favor atheroma stenosis versus aneurysms. Th1/Th2 cytokine balance is implicated in transplant studies with aortic allografts (Shimizu et al, 2006), whereas hyperlipi- demia increases aneurysms in humans (Wanhainen et al, 2005) and in a mouse model (Zhao et al, 2004). Of great importance, arterial aging begins before birth in ‘prodromal lesions’ (Davies, 1990; Hirsch, 1941; Hirvonen et al, 1985; Leistikow, 1998; Napoli et al, 1999; Palinski and Napoli, 2002; Stary, 2000). Fetal arteries have microscopic cell clusters of macrophages and oxidized LDL that may be seeds of adult plaques (D’Armiento et al, 2001; Napoli et al, 1999; Sims, 2000). The mass of oxidized LDL, macrophages, and MMP-9 increases linearly in the aorta and common carotid, but may accelerate exponentially in intra-cerebral arteries (Fig. 1.15B). Between age 10 and 20 y, intimal layer macrophage density increased pro- gressively in coronary arteries (Sims et al, 2002). By early adult life, advanced arterial lesions are common. An early glimpse of this came from autopsies of sol- diers killed during the Korean and Vietnam Wars. Many of these healthy young men had coronary artery degeneration, nearly one-third had >50% narrowing (Joseph et al, 1993; McNamara et al, 1971). These findings are confirmed by large multi-ethnic autopsy samples: PDAY Study (Pathobiological Determinants of Atherosclerosis in Youth) (Strong et al, 1999) and Bogalusa Heart Study (Berenson, 2004; Li et al, 2003). By age 30, about 50% of men and 35% of women have raised coronary lesions (Strong et al, 1999).
  • 74 The Biology of Human Longevity Lipids accumulate faster in children exposed to maternal hypercholesterolemia during pregnancy (FELIC Study, Fate of Early Lesions in Children) (Napoli et al, 1999) (Section 4.8). The variability of lesion size increased during later childhood, implying influences include diet and exercise (Chapter 3). Obesity may accelerate coronary atherosclerosis in young men more than in women (McGill et al, 2002) (Chapter 3). The greater arterial degeneration in men corresponds to the strong sex biases in cardiac disease and mortality (Tuzcu et al, 2001) (Section 2.10.4). The fate of the prodromal fatty streaks is not fixed. Fatty streaks, while ‘clini- cally silent,’ may regress or develop further into advanced plaques that are asso- ciated with occlusive vascular disease. The transience of fatty deposits in neonatal aortas is well known (Hirsch, 1941). Thus, the level of atherogenesis in early life may not predict advanced lesions later in life (Madsen et al, 2003; Stary et al, 1994). In adults, advanced atheromas may regress during stain treatment (Petronio et al, 2005) (Chapter 2) or certain wasting conditions (Eilersen and Faber, 1960) (Chapter 3). 1.5.2. Hazards of Hypertension The central arteries develop as highly elastic reservoirs for the blood volume expelled at each heartbeat. In children, the aorta literally balloons at each heart- beat in response to the force of the pulse wave. By puberty, arterial thickening and stiffening begin to increase the pulse wave velocity and blood pressure into the adult range, >100 mm systolic pressure measured on the arm (‘cuff,’ or brachial pressure) (Fig. 1.6B). Progressive increases of systolic pressure after age 30, about 0.7 mm systolic pressure/year, soon enough depart increasingly from the clinical goal of <120 mm Hg systolic and <80 mm diastolic6 (O’Rourke and Nichols, 2005). These age findings were established by the Framingham Study, a pioneering community-based study, and are generalized to aging pop- ulations worldwide. By age 80, average pulse wave velocity has doubled and the systolic pressure has crept up to about 140 mm; this degree of elevation was for- merly considered ‘border-line hypertension,’ but is now considered risky and warranting intervention. Worse, pulse pressure in the aorta increases with aging are greater than the brachial systolic pressure, up to 4-fold (O’Rourke et al, 2004; O’Rourke and Nichols, 2005; Safar, 2005). 6 The Sixth Report of the National Committee on Detection, Evaluation, and Treatment of High Blood Pressure (Anonymous, 1997a). Systolic pressure (brachial, or cuff) is accepted as a stronger risk indicator of vascular complications than diastolic pressure; shown by Framingham and confirmed by meta-analysis of two huge data assemblages (Black, 2004). Pressure wave reflections (‘augmentation index’) of the central pulse wave form give more information about vascular disease than cuff pressure. Brain and kidney arteries are more exposed to this higher pressure than other tissues (O’Rourke and Nichols, 2005).
  • Uncontrolled systolic hypertension increases the risk of stroke, heart attack, congestive heart failure, and kidney failure. These hazards operate even in the supposedly normal range of systolic blood pressures. Vascular mortality risks from ischemic heart disease and stroke increase exponentially with blood pressures above 115/75 mm Hg; the curves are strongly age stratified. The Framingham Study concluded “cutoff points to define ...hypertension are arbi- trary” (Sagie et al, 1993). The reality of these hazards is confirmed by the bene- fits of drug treatments, which lowered cardiovascular events, congestive heart failure, and stroke by about 30% during 4.5 y (SHEP, Systolic Hypertension in the Elderly Program) (Perry et al, 2000). Stroke incidence was decreased by about 1% for every 1 mm of lowered systolic pressure. However, at any systolic pres- sure, age increases mortality risks, by about 50-fold from 40–89 years (meta-analysis, 61 prospective studies) (Lewington et al, 2002) (Fig. 1.6C). Even modest increases of systolic pressure increase the risk of subsequently developing clinical hypertension. Systolic pressures of >160 mm increase to a prevalence of about 30% by age 60 y in most populations. The risk of hyper- tension is predicted by basal elevation years before. Even those with mild elevations (131 mm average) had a 50% higher risk of hypertension (Franklin, 2005; Franklin et al, 2005). Hypertension has the malignant feature of causing further arterial wall thickening. Hypertension can synergize with hyperlipidemias, particularly elevated LDL cholesterol. Carotid IMT increased with blood pressure, except when LDL cho- lesterol was low (Los Angeles Atherosclerosis Study) (Sun et al, 2000). Thus, ele- vated systolic blood pressure, which increases in prevalence with age (Fig. 1.6A), appears to increase arterial susceptibility to damage from LDL cholesterol. Synergies of hypertension and hyperlipidemias are shown in animal models (below, 1.6.4). Hyperlipidemias and inflammatory factors are discussed in 1.6.4. Other blood indicators are discussed in 1.5.4. 1.5.3. Mechanisms Arterial aging changes and atherogenesis appear to share inflammatory process that are, at least in part, driven by the physics of blood flow. Flow characteris- tics operate on arteries in two modes: in signal transduction through flow sensitive membrane links to the cytoskeleton and by direct force on the rigid body of atheromas. Inflammatory processes that participate in both modes may also interface with external infections and inflammogens (Chapter 2). 1.5.3.1. Inflammation For nearly two centuries, inflammation has been a suspected cause of vascular dis- ease, from Virchow to Ross, who emphasized that processes in atherogenesis are shared with other major chronic inflammatory diseases, including pulmonary fibro- sis, rheumatoid arthritis, and renal glomerulosclerosis. Synergies of hyperlipidemia Inflammation and Oxidation in Aging and Chronic Diseases 75
  • 76 The Biology of Human Longevity and hypertension illustrate Ross’s hypothesis that atherosclerosis is an inflammatory ‘response to injury.’ True to Celsus’s classic signs of inflammation (calor, or heat), atheromas are hotter than flanking vascular tissues, e.g., by 1˚C in a rabbit model (Verheye et al, 2002). Currently, inflammation can be both cause and effect in arte- rial disease (Tracy, 2002). As described below, hemodynamics has a major role in both the arterial thickening of usual aging and atheroma formation, with funda- mental involvement of inflammatory processes. Inflammatory processes of innate immunity are at the core of atherogenesis. Lipid accumulation by macrophages, particularly oxidized LDL, is mediated by scavenger receptors (SR-A, CD-36) (Ricci et al, 2004; Shashkin et al, 2005), lead- ing to the characteristic ‘foam cells’ of atheromas. Foam cells secrete cytokines and chemoattractants that activate smooth muscle cells. Cell growth factors (MCSF) and cell adhesion factors (VCAM1) mediate attachment of macrophages and platelets (Cunningham and Gottlieb, 2005; Dai et al, 2004; Passerini et al, 2004). Toll-family receptors (TLRs) mediate cytokine secretion, including TLR 4, which also binds LPS endotoxin (Miller et al, 2005). During atherogenesis, many inflammatory proteins are produced by endothelial cells, smooth muscle cells, and macrophages (Table 1.3). The growing list includes cytokines (IL-1, IL-6, IL-8, TNFα) and complement (C) factors (C1q, C1r, C3, C5). Many of these genes are regulated by NF-κB family transcription factors, which are redox sensitive and mediate gene regulation during inflammation and oxidative stress (Li et al, 2002; Li et al, 2005b; Monaco and Paleolog, 2004). NF- κB increases in smooth muscle cells of atheromas relative to adjacent normal arterial areas (Bourcier et al, 1997; Hajra, 2000). Many complement proteins are activated in atheromas, but fewer complement inhibitors are found (Yasojima et al, 2001a,b). Cell death in arterial plaques is closely associated with the terminal complement membrane attack complex, C5b-9 (Niculescu et al, 2004). The anaphylactic peptide C5a is also produced during com- plement activation and is a potent chemoattractant and activator of macrophages, causing release of TNFα and reactive oxygen species that cause further oxidative damage (Query I and II). Plasma elevations of C5a may be a risk factor in cardio- vascular events (Speidl et al, 2005) together with CRP (Fig. 1.16). CRP can also acti- vate the complement system (Pepys and Hirschfield, 2003). Both CRP and complement factors are produced by plaque smooth muscle and macrophages, more than in normal arteries (Jabs et al, 2003; Yasojima et al, 2001a,b). CRP at acute phase levels can increase LDL uptake by macrophages (Fu and Borensztajn, 2002; Zwaka et al, 2001) through inducing the receptor for oxidized LDL (LOX-1) (Li et al, 2004). PTX3, an anti-microbial pentraxin related to CRP can be made by macrophages and vascular smooth muscle cells, and is increased by oxidized LDL (Klouche et al, 2004; Rolph et al, 2002). Systemic CRP and fibrino- gen enhance macrophage accumulation in plaques. Moreover, CRP elevations may influence T cell responses of adaptive immunity, through inhibiting dendritic cell differentiation (Zhang et al, 2006). Subsets of the inflammatory proteins of atheromas also occur in senile plaques of Alzheimer disease (discussed below).
  • Arterial amyloid accumulations may be universal by middle age (Schwartz, 1970) and can include a variety of proteins (Buxbaum, 2004; Kawamura et al, 1995). SAA (serum amyloid A) and SAP, both acute phase proteins,7 are common in atheromas (Li et al, 1995; O’Brien et al, 2006; Yamada et al, 1996). Elevated plasma SAA is a coronary risk factor (Yamada et al, 1996) and is a source of the SAA in aging arter- ies. By binding to HDL during the acute phase response, SAA impairs HDL’s anti- oxidative protection to LDL and becomes ‘proinflammatory’; SAA/LDL complexes are more oxidized than native LDL and are coronary risk factors (Chait et al, 2005; Ogasawara et al, 2004; Van Lenten et al, 1995). Another amyloid protein, medin- amyloid, is common by age 60 in the aorta (Peng et al, 2002; Peng et al, 2005a,b). Medin, a peptide fragment of lactadherin, is made by smooth muscle cells and is Inflammation and Oxidation in Aging and Chronic Diseases 77 RelativeRisk 159 A 127 0.93 1.58LDL-cholesterol, mg/dl IL-6, pg/ml *** *** ** ** * * 2 4 6 8 10 12 0 1.3 1.1 1.0 2.8 2.5 1.2 4.4 3.4 2.8 Total: HDL Cholesterol Ratio C-Reactive Protein High Medium Low B Low Medium High RelativeRisk FIGURE 1.16 Additivity of blood inflammatory and lipid risk markers for cardiovascular events. A. IL-6 and LDL cholesterol in the PRIME study (Prospective Epidemiological Study of Myocardial Infarction; population based sample of 10,600 men, 50 to 59 y from France and Ireland. IL-6 also correlated with CRP and fibrinogen. (Redrawn from Luc et al, 2003.) B. C-reactive protein, HDL, and risk of first myocardial infarction (MI) in the Physicians’ Health Study of apparently healthy men (ran- domized study of 22,071 middle-aged U.S. male physicians, 95% Caucasian without prior MI, stroke, TIA, or cancer at the start. (Redrawn from Ridker et al, 1998.) 7 SAP is an acute phase reactant in mice, but not humans, whereas SAA is an acute phase reactant in both species.
  • deposited on the elastic lamina, where it may facilitate macrophage binding; lactadherin inhibits rotavirus infections and participates in angiogenesis. Atheromas accumulate advanced glycation end products (AGEs), which are hypothesized to accelerate lipid oxidation, as is observed in diabetics (Reaven et al, 1997) (bystander damage, type 1 and 2, Section 1.4.1). Blockade of the receptor for AGE (RAGE) by a soluble truncated protein (sRAGE ligand) suppressed atherosclerosis in a mouse model with a 10-fold dose response (Naka et al, 2004). Conversely, endothelial RAGE overexpression enhanced diabetic damage to retina and kidney (Yonekura et al, 2005). Increased formation of superoxide (O2 – ) is characteristic of endothelial dys- functions in arterial disease and is attributable to increased NAD(P)H oxidase activity (Guzik et al, 2006). The NAD (P)H oxidase subunit p22phox is 50% higher in diseased than healthy coronary arteries, in correlation with inflammatory cell content. Consequences include quenching of nitric oxide (NO) and activation of redox pathways that promote remodeling. The importance of superoxide to arte- rial disease is indicated by benefits to endothelial functions in rodent models of increased superoxide dismutase by gene transfer (Longo et al, 2000). Iron accumulates as a bystander process of oxidative damage, as well as causes bystander damage (Section 1.4.1). Oxidized LDL cholesterol (oxysterols) is associated with depots of intracellular ferritin in macrophages and foam cells (Li et al, 2005d; Stadler et al, 2004). Superoxide also releases iron from aconitase (Fe-S complex) (Longo et al, 2000). Iron (Fe2+ ), in turn, causes oxidative injury from the hydroxy radical (Fenton/Haber-Weiss reaction) (Fig. 1.11) leading to cell death. Necrosis in the plaque core increases risk of plaque rupture. Iron chelators decreased caspase induction and apoptosis in animals (Li et al, 2005d). Frequent human blood donors had about 40% fewer cardiovascular events (Meyers et al, 2002), consistent with hypotheses that lower body iron is protec- tive by decreased oxidation of low-density lipoprotein cholesterol (Meyers et al, 2002) and lowered intracellular soluble iron (Li et al, 2005d). A further outcome is the calcification of atheromas (Fig. 1.13, stage VII), a focal calcification differing from the more universal and diffuse arterial elastocalcinosis (Dao et al, 2005). Calcification is associated with cell death (apoptosis) of vascu- lar smooth muscle and accumulations of monocytes, distinct from foam cells, mast cells, and oxidized lipids (Jeziorska et al, 1998; Tintut et al, 2002). Medial layer cells can differentiate as osteoblasts to form calcium phosphate crystals resem- bling bone mineral (Abedin et al, 2004). Neovascularization also stimulates ectopic calcification (Collett and Carfield, 2005). Calcification is an independent risk factor in vascular mortality (Doherty et al, 2003; Sangiorgi et al, 1998). Lastly, and of possible major importance, B and T lymphocytes are common in atheromas. A recent study showed 10-fold increases of T cells in unstable atheromas with increased INFγ and IL-4 (De Palma et al, 2006). T cell spec- tratyping showed oligoclonal expansions, suggesting antigen-driven recruitment, differing between plaques of individuals, which could be critical to atheroma instability and cell lysis. The elevated plasma CRP that predicts vascular events 78 The Biology of Human Longevity
  • may sensitize endothelial cells to T cell cytotoxicity. (Nakajima et al, 2002). Ubiquitous CMV infections, which are implicated in the increase of highly dif- ferentiated T cells during immunosenescence (Section 1.2, Sections 2.2 and 2.7) could increase autoimmune interactions leading to plaque instability. 1.5.3.2. Hemodynamics As noted above, atheromas tend to form at arterial branches and along inner curves, where the vascular geometry slows blood flow and produces low shear forces (Moore et al, 1994; Traub and Berk, 1998; Wootton and Ku, 1999). Later, as plaques protrude into the lumen, they are increasingly exposed to increased tensile stress on the plaque wall, which may cause fissures and fractures (Slageret et al, 2005a,b). Thus, normal blood flow may cause plaque instability through mechanical forces. Signal transduction is the other major mode of hemodynamic influences on arterial functions. Blood flow directly regulates inflammatory responses through mechanosensors in endothelia (Li, 2005). The role of blood flow in the progres- sion of plaques is shown in elegant studies that combine hemodynamic models and molecular biology (Cunningham and Gotlieb, 2005; Dai et al, 2004; Pfister et al, 2005). Flow force transduction is thought to be mediated by endothelial cell cytoskeleton through links, which include G proteins, ion channels, integrins, PDFG-receptors, etc. Downstream mechanisms include activation of machinery shared with inflammatory responses including NF-κB transcription factors and Akt kinases (Kakisis et al, 2005; Li, 2005). Another inflammation-related mechanism is the sphingomyelinase in caveola of endothelial cells, which is flow-activated and produces transients of ceramide (Czarny and Schnitzer, 2004), a long-chain fatty acid. Ceramide mimicked the flow-activation of eNOS, which produces NO, a key vasodilator and endothelial cell regulator that is at lower levels in atheromas than healthy endothelia (Cunningham and Gotlieb, 2005; Wilcox et al, 1997). Furthermore, sphingolipid and eicosinoid pathways have multiple synergies in inflammatory responses (Pettus et al, 2005) that may mediate the increased production of prostaglandins by macrophages during aging (Wu, 2004). Distinct arterial waveforms, ‘atheroprone’ and ‘athero-protective,’ cause differ- ent patterns of gene expression in the vascular endothelia (Dai et al, 2004). Atheroprone flow induced a number of proinflammatory genes (IL-1, IL-6, IL-8; tumor necrosis factor receptor superfamily, member 21 [TNFRSF21]; complement C1r and C3; and PTX3), but decreased expression of IL-10, an anti-inflammatory cytokine (Dai et al, 2004; Passerini et al, 2004). NF-κB was 4-fold higher in endothelial cells of atheroprone regions, consistent with activation of inflamma- tory genes (Hajra et al, 2000). In contrast, normal arterial flow represses arterial inflammatory gene expression, e.g., VCAM1, which mediates cell adhesion, via a TNF-dependent pathway (Yamawaki et al, 2003; Yamawaki et al, 2005). Atheromas accumulate replicatively senescent endothelial cells above normal aging arteries (Minamino et al, 2004). Telomere DNA loss in vascular endothelial Inflammation and Oxidation in Aging and Chronic Diseases 79
  • cells correlates with the grade of atheroma (Edo and Andres, 2005; Okuda et al, 2000). Replicatively senescent endothelial cells have lower eNOS, a key regula- tion of vasodilation noted above (McCarty, 2004). A loss of NO production would decrease the protection of endothelial cells from apoptosis in response to oxi- dized LDL and hyperglycemia. The declining production of NO during cell senes- cence is blocked by maintaining telomere length by transfection with hTERT, the catalytic unit of telomerase (Minamino et al, 2002). Moreover, telomerase in endothelial progenitor cells is inhibited by angiotensin II (Ang II), which induces oxygen-derived free radicals through gp91phox and other subunits of the vari- ous NAD(P)H oxidase complexes. The inhibition of telomerase by Ang II was blocked by SOD, or by an angiotensin receptor (AT1) antagonists (Imanishi et al, 2005). These findings epitomize the multiple interactions of oxidative damage and inflammation across multiple levels of molecular, cellular, and physiological organization in atherogenesis. I suggest that endothelial cell senescence represents bystander damage from proliferation stimulated by blood pulsation and inflammation (bystander types 3 and 4, Section 1.4). The weaker correlations of telomere loss with age than grade of atheroma (Okuda et al, 2000) support bystander mechanisms in the inflammatory milieu of the atheroma. Moreover, shortened telomeres in circulat- ing white blood cells of hypertensives were correlated with carotid artery plaque load, possibly reflecting systemic inflammation (Benetos et al, 2004). Endothelial cell senescence is linked to the insulin/IGF-1 pathways implicated in vascular disease and in longevity (Fig. 1.3). During serial endothelial culture leading to replicative senescence, Akt kinase levels increased 4-fold, which is con- sistent with the arrest of growth in early passage by activation of Akt (Miyauchi et al, 2004). Akt activation inhibits FOXO3, a transcription factor orthologue of DAF-16 in worms that controls transcription of MnSOD and other anti-oxidant genes (Fig. 1.3A). Thus, Akt activation in atheromas could increase oxidant stress by diminishing the capacity to remove reactive oxygen species. The IGF-1 path- way is a target of drug development for vascular disease (de Nigris et al, 2006). Besides their sensitivity to flow-induced inflammatory gene expression, atheroprone arterial regions show greater sensitivity to LPS, the endotoxin of common infections: In a mouse model, LPS caused greater induction of the NFkappaB in atheroprone than in resistant regions (Hajra et al, 2000). Moreover, the atheroprone regions had greater induction of VCAM and E-selectin, proatherogenic genes that use NF-κB. Inflammation in atherogenesis is greater after priming by LPS and possibly during infections (Chapter 2). Blood flow regulates nitric oxide (NO), a key vasodilator that may be protec- tive in atherosclerosis by inhibiting apoptosis, platelet aggregation, smooth muscle proliferation, and white blood cell adhesion apoptosis (Tai et al, 2005). Free NO from endothelial nitric oxide synthase (eNOS) reacts with superoxide to form peroxynitrite and other reactive oxygen species (ROS) (Fig. 1.11), which can cause molecular damage (Cai, 2005; Heck et al, 2005). The increased levels of protein nitration in atheromas and in aging arteries independently of atheromas 80 The Biology of Human Longevity
  • (e.g., >2-fold) are attributed to NO-derived oxidants (van der Loo et al, 2000). The increased production of superoxide in aging arterial walls and atheromas is hypothesized as an important source of oxidative damage. Hypertension is strongly associated with increased arterial wall thickening, atherosclerosis, and vascular mortality in humans (see above). Animal models document these associations (Lerman et al, 2005). A transgenic hypertensive rat with increased angiotensin II had thicker aortic walls with more smooth muscle cells and collagen (Rossi et al, 2002). However, hypertension-induced changes may not progress to atherosclerotic plaques without hyperlipidemia. Moreover, hypertension interacts with hyperlipidemia. In the hyperlipidemic Watanabe rabbit model, 3 months of induced hypertension caused a 4-fold greater increase of aortic lesions (Chobanian et al, 1989). Increased synthesis of collagen and tropoelasin are implicated in the aortic compensatory responses to tensile stress from hypertension (Xu et al, 2000; Xu et al, 2002). Synergies in the age-related increase of hypertension and hyperlipidemia could accelerate mortality from vascular causes (see Section 1.5.3.3). Lastly, the loss of arterial elasticity during arterial aging could also be hemo- dynamically driven. According to O’Rourke’s hypothesis (O’Rourke and Nichols, 2005), elastin fibers fragment from fatigue during repeated stress cycles. So far, elastin has not been characterized for fatigue and fracture characteristics. Using natural rubber as a model and assuming 10% stretch per cycle, as in the thoracic aorta, elastin fractures might emerge by 800 million cycles (heartbeats) in the third decade (O’Rourke, 1976), which is at the onset of stiffening and elevations of systolic pressure (Fig. 1.6A). Alternatively, I suggest that elastin degradation is enzymatically caused by endoproteinases (MMP-2) as were localized to sites of elastic lamina fragmentation in aging rat aortas (Li et al, 1999). Elastin fiber fatigue might increase protease susceptibility. Both mechanisms are consistent with accelerated loss of elasticity in hypertension. 1.5.3.3. Aging The vascular aging changes in arterial wall thickening and atherogenesis are very slow, indolent processes that typically take decades before adverse impact. Edward Lakatta and colleagues propose that ‘aging’ is a key parameter in the pro- gression of arterial disease (Lakatta, 2003; Lakatta and Levy 2003a,b; Wang and Lakatta, 2006). “[Vascular aging changes] . . . create a metabolically and enzy- matically active milieu . . . conducive for superimposed atherosclerosis. An important corollary is that age should no longer be viewed as an immutable car- diovascular risk factor.” (Najjar et al, 2005, p. 460). The consideration of vascu- lar disease in terms of ‘(aging process) × (exposure time)’ (Lakatta, 2003) joins the discussion of bystander/event-related aging (Section 1.4). A shift in thinking is underway which challenges the assumption that aging is an immutable process. In an emerging view, the rate of arterial aging processes is dependent on multiple interactions in the internal and external milieu of the individual. Inflammation and Oxidation in Aging and Chronic Diseases 81
  • 82 The Biology of Human Longevity There is extensive overlap between arterial age changes and the cell processes in atherogenesis. Arterial aging processes are broadly shared in rodents, rabbits, monkeys, and humans (Table 1.4). Thus, vascular aging processes join brain amyloid accumulation and ovarian oocyte loss in the mammalian canon of aging. Species vary in the extent of particular changes and in the proclivity to athero- genesis in response to diet and stress. Wall thickening and atheroma formation both share many molecular and cellular changes that are broadly considered inflammatory processes (D’Armiento et al, 2001; Najjar et al, 2005). Both have subclinical phases beginning early in life: Prodromal atheromas appear to begin even before birth in the form of minute cell foci that may develop later into life- threatening atheromas. Vascular aging and atherogenesis involve local cell pro- liferation, invasion of macrophages, and excess production of collagen. Aging increases the permeability to albumin and other blood proteins (Section 1.6.3). As also noted, aging arteries have elevated cytokines (TNFα) and genes associ- ated with apoptosis (caspases), chemotaxis (MCP-1), and matrix remodeling (MMP-2). Senescence of endothelial progenitor cells (section above) impaired proliferation and migration (Heiss et al, 2005). In old rat arteries, smooth muscle cells secrete more proinflammatory cytokines, whereas old endothelial cells secrete more of the prothrombotic PAI-1 (plasminogen activator inhibitor-1); production of vasodilators (NO, prostacyclin) decreases, whereas vasoconstric- tors increase (angiotensin II and endothelin) (Najjar et al, 2005). Moreover, the TABLE 1.4 Comparisons of Arterial Aging Changes in Humans and Mammalian Models with Atherosclerosis and Hypertension Arterial Parameter Human Monkey Rat Rabbit Atherosclerosis Hypertension Diffuse intimal thickening + + + + + + Lipid deposits − − − − + +/− Macrophages + − − − + + T cells + − − + + + Matrix ↑ 0 + + + + + Ang II-ACE ↑ + + + + + + Endothelial dysfunction + + + + + + Extra cell. Matrix + + + ? + + ICAM ↑ ? ? + ? + + MCP-1/CCR2 ↑ + + + + + + NADPH oxidase ↑ ? ? + ? + + TGF-β1 ? + + ? + + VEGF ↑ + ? ? + + + Lumenal dilation + + + + ? +/− Wall stiffness ↑ + + + + ? + Collagen ↑ + + + + ? +/− Elastin degeneration + + + + 0 0 Telomere shortening + + + ? + ? Adapted from Lakatta (2003), Lakatta and Levy (2003a,b), Wang and Lakatta (2006), Najjar et al (2005).
  • interactions of hypertension, which increases sharply during aging, could syner- gize with hyperlipidemia (Chobanian et al, 1989; Sun et al, 2000; Xu et al, 2000; Xu et al, 2002) (Section 1.6.3) in the accelerating vascular mortality. Primary cultures of vascular cells also show major donor age effects consis- tent with increased atherogenesis. Human vascular smooth muscle cell cultures showed progressive decline in proliferation and migration from donors across adult ages (Ruiz-Torres et al, 2003). Stimulation of proliferation by insulin and IGF-1 declines with donor age (Ruiz-Torres et al, 1999; Ruiz-Torres et al, 2005). Endothelial lines derived from two donors aged 36 and 90 years (hmEC36 and hmEC90, respectively) differed in the capacity for new blood vessel formation on collagen-gels (Koike et al, 2003): the younger hmEC36 supported much more angiogenesis. Proteases critical for angiogenesis differed correspondingly: Active MMP-2 was produced only by hmEC36, whereas hmEC90 produced much more protease inhibitor (TIMP-2). Cultured human vascular endothelial cells also pro- gressively lose telomere DNA (Chang and Harley, 1995). Rodent models show cell aging changes. Smooth muscle cells from old rats secrete more proinflammatory cytokines, whereas old endothelia secrete more of the prothrombotic PAI-1 (plasminogen activator inhibitor-1); production of vasodila- tors (NO, prostacyclin) decreases, whereas production of vasoconstrictors increases (angiotensin II and endothelin) (Najjar et al, 2005). Smooth muscle cells from 2-year-old rats had greater induction of MMP-2 production in response to IL-1α and TNFα (Li et al, 1999), which could enhance elastin degradation, as noted above. Aging may increase endothelial responses to mechanical injury: Older rats had much greater myo-intimal proliferation in response to ‘de-endothelialization’ by wire scrape than young adults (Hariri, 1986). This elegant study showed that the age effect persisted when old aortic segments were transplanted to young aortas. No generalizations are at hand, because other experimental models present different age changes in vascular proliferative responses (Torella et al, 2004). These finding suggest that aging arteries are more sensitive to atherogenic stimuli or injury. Clearly, multiple processes are at work in vascular aging and atherogenesis across the life span. These and many other arterial changes address Query I of bystander oxidative damage promoting inflammation and Query II of inflammation causing further bystander damage. The co-occurrence of macrophages and oxidized lipids in fetal arteries exemplifies the fetal origins of adult arterial disease (Chapter 4) and points to specific and fundamental roles of oxidant damage and inflammation in arterial aging. These hypothesized interactions of oxidized lipids and inflammation are directly tested by transgenic over-expression catalase in mice (Section 1.2.6), which attenuated arteriolosclerosis and myocardial calcification and fibrosis in ad lib feeding (Schriner et al, 2005) and in a hyperlipidemic model of accelerated atherosclerosis attenuated the size of arterial lesions and plasma and arterial lipid peroxidation (Yang, 2004a). Thus, the pathogenic interactions in Fig. 1.2A,B are ongoing processes throughout the life history. We must also recognize effects of the progressive increase in systolic pressure during aging on endothelial changes, which are expected to be compounded by inflammation and obesity (Chapters 2 Inflammation and Oxidation in Aging and Chronic Diseases 83
  • and 3). We may anticipate a detailed theory that accounts for accelerating mor- tality during aging from hemodynamics, which incorporates microscopic and macroscopic vascular changes to predict population mortality risks. 1.5.3.4. Endothelial Progenitor Cells Circulating endothelial progenitor cells (EPCs) are increasingly implicated in vas- cular disease by roles in plaque repair (endothelialization) and neovascularization of ischemic tissue (Dong et al, 2005; Urbich and Dimmeler, 2005). Adults maintain circulating EPCs (CD34+ , CD133+ ) that are derived from the bone marrow. EPCs may ‘home’ to sites of ischemia and enhance in vascular repair and angiogenesis. EPC numbers varied inversely with total cholesterol and LDL cholesterol (Chen et al, 2004) and other clinical atherosclerotic risk factors (Vasa et al, 2001; Steiner et al, 2005), and were 40% lower in a sample of coronary patients (Vasa et al, 2001). Although EPC numbers did not show age declines, there may be age decreases in proliferation and migration (Heiss et al, 2005). These deficits correlated with brachial flow-mediated dilation, a measure of endothelial dysfunction. Rodent models also showed EPC deficits in hypercholesterolemia and the ben- efits of supplemental EPCs. In hypercholesterolemic mice (apoE−/−), arterial fatty deposits were diminished by infusion of EPCs from normal mice, whereas fewer EPCs were obtained from 6-month-old apoE−/− with advancing atherosclerosis (Rauscher et al, 2003). The old-derived EPC induced patterns of arterial wall gene expression that resembled gene expression in advanced lesions in mice and in humans (Karra et al, 2005). Thus may bone marrow aging contribute to athero- genesis. Moreover, EPCs can transdifferentiate into myocardial lineage cells that contribute to myocardial regeneration (Murasawa et al, 2005). The EPC capacity for neovascularization of ischemic tissue may depend on their unusual resistance to oxidant stress through elevated MnSOD (He et al, 2004). Endothelial cell replicative senescence may decrease the pool of EPCs that repair vascular damage and increase risk of atheroma rupture. Statins may also modulate EPC cell senescence by suppressing Chk2, a DNA damage checkpoint kinase that is induced by telomere dysfunction (Spyridopoulos et al, 2004). Inflammation and smoking also inhibit EPC functions (Section 2.5.2). To antic- ipate this subsequent discussion, a specific inflammatory class of endothelial cells (IEPs) was recently described. (Holmen et al, 2005). Circulating IEPs have higher expression of inflammatory markers, which inhibits their functions. Blood levels of IEC correlated with serum CRP in vasculitis patients. 1.5.4. Blood Risk Factors for Vascular Disease and Overlap with Acute Phase Responses The blood indicators of vascular disease include several acute phase reactants in response to infections, e.g., IL-1, IL-6, and CRP elevations (Section 1.3). Moreover, the combination of elevated triglycerides and LDL- and low HDL-cholesterol that 84 The Biology of Human Longevity
  • are risk indicators of vascular events (Braunwald, 1997; Castelli, 1996) is also observed during infections. In this brief review of a huge field, vascular event predictions improve by includ- ing other markers, particularly of inflammatory proteins, in addition to LDL or HDL cholesterol (Fig. 1.16). Plasma elevations of CRP and IL-6 are separately and together considered as risk indicators for a first-ever or recurrent vascular event. Even modest elevations of CRP (top two tertiles) increase risks of the first or recur- rent heart attacks. The strongest predictor of vascular events was elevated CRP in combination with lower cholesterol: HDL cholesterol ratio [Physicians Health Study (Libby and Ridker, 2004) and Women’s Health Study (Ridker et al, 2000). Even after adjusting for cholesterol, high CRP predicts stroke (Rost et al, 2001)]. However, IL-6 associations may be as strong as CRP. In the PRIME Study (Prospective Epidemiological Study of Myocardial Infarction) of healthy middle age, IL-6 was the strongest predictor (Luc et al, 2003): The highest tertile of CRP, fibrinogen, IL-6, and LDL cholesterol were each associated with 2–3-fold higher risk; however, the best predictor was IL-6 in combination with LDL cholesterol. Other vascular risk mark- ers include fibrinogen, homocysteine, Lp(a), plasminogen activator inhibitor (PAI-1), and TNFα. For a balanced view of the complex contending statistical argu- ments on CRP and other risk factors, see Davey Smith et al. (2006). A substantial portion of sporadic events occur within the normal range of lipid risk factors, e.g., 35% of heart attacks in the original Framingham sample (Castelli, 1996). High CRP predicted stroke, after adjustment for total and HDL cholesterol (Rea et al, 2005). Infections may be unidentified risk factors lurking in these large population studies, because acute infections induce many of the same dyslipidemias and other acute phase changes shared with vascular risk fac- tors (Esteve et al, 2005; Khovidhunkit et al, 2004; Ohsuzu, 2004). Blood triglyc- erides increase during acute infections, as energy is mobilized by lipolysis. Concurrently, infections decrease HDL and the ‘reversed cholesterol transport’.8 Both shifts are vascular risk factors. The same changes are induced by bacterial endotoxins, which cause similar remodeling of HDL and LDL particles. Acute infections also increase the oxidation of blood LDL and VLDL, the uptake of oxi- dized lipids by macrophages, and the inhibition of reversed cholesterol transport, which removes cellular cholesterol to the liver for recycling. During the acute phase, HDL is remodeled into the proinflammatory ‘acute phase HDL’ in complex and incompletely understood mechanisms (Ansell et al, 2003; Khovidhunkit et al, 2004; Van Lenten et al, 2006). Normal HDL particles have anti-oxidant activities that protect LDL from oxidation (Getz and Reardon, 2004; Kontush et al, 2003) and that inhibit LDL-induced monocyte chemotactic responses (Ansell et al, 2003). Normal HDL also blocks some proinflammatory activities of CRP (Wadham et al, 2004). Inflammation and Oxidation in Aging and Chronic Diseases 85 8 Total cholesterol and LDL differ in response to infections by species with increases in rodents and rabbits, but decreases in humans and primates.
  • Many infections impair the protective effects of HDL. The ‘acute phase HDL’ has fewer anti-oxidant activities, due to the loss of antioxidant proteins, trans- ferrin, and the paraoxinases (PON-1, PON-3), which hydrolyze oxidized lipids in LDL (Van Lenten et al, 2006). The acute phase remodeling involves binding of LPS to the LPS-binding protein (LBP) and the phospholipid transfer protein (PLTP), which normally regulates the transfer of phospholipids from cell mem- branes to HDL (Levels et al, 2005). LPB and PLTP are acute phase responses and may be directly bacteriocidal because of their structural similarities to lipid trans- fer proteins that increase bacterial permeability (Kirschning et al, 1997). Acute phase elevations of serum amyloid A (SAA) displace the apoA-I on HDL, which impairs reversed cholesterol transport (Miida et al, 2006). SAA also enhances cholesterol uptake by macrophages. The Toll-receptor pathways that mediate responses to infections are also implicated in atherogenesis (Bjorkbacka et al, 2004). Cholesterol efflux from macrophages is mediated by cross-talk between Toll-like receptors on macrophages and LXR receptors (the nuclear receptor liver X receptor) (Castrillo et al, 2003). Toll-activation by infections thus could contribute to atherogenesis by favoring the accumulation of cholesterol in arterial macrophages. The LXR pathway is emerging as a key nexus of innate immunity in protection of macrophage apoptosis (Valledor, 2005) The macrophage scavenger receptor (MSR-A), which binds viral and bacterial pathogens, also mediates the uptake of oxidized lipoproteins (Gordon, 2003; Suzuki et al, 1997). Two other inflammatory changes could enhance lipoprotein uptake by vas- cular macrophages: Infections tend to oxidize lipoproteins and release ceramide and sphingomyelin (Auge et al, 2002; Hajjar, 2000) and the increased CRP, as noted above (Fu and Borensztajn, 2002; Zwaka et al, 2001). Hajjar and Haberland (1997) describe the uptake of oxidized LDL by vascular macrophages as a ‘molecular Trojan horse’ by inducing inflammatory processes that contribute to atherogenesis. The increased ceramide and sphingomyelin from lipoprotein oxi- dation during infections also stimulate proliferation of vascular smooth muscle cells (Auge et al, 2002). Infections in vascular disease are discussed in Chapter 2. This brief review of a huge literature shows the profound involvement of inflammation in arterial aging. Modulations of arterial aging by systemic inflam- mation and pathogens (Chapter 2) and by diet (Chapter 3) will draw on the ele- ments discussed above. Many of these same factors are also central to Alzheimer disease. 1.6. ALZHEIMER DISEASE AND VASCULAR-RELATED DEMENTIAS Unlike heart attacks, Alzheimer disease and vascular-related dementias are rare (<1%) before age 65. Alzheimer disease prevalence accelerates with a doubling of risk every 5 y (Fig. 1.10A). The pathologic markers of Alzheimer disease 86 The Biology of Human Longevity
  • are senile plaques (extracellular ‘neuritic plaques’), neurofibrillary tangles (intra- neuronal ‘tangles’) (Fig. 1.10B), and selective neuron loss. Tangles and plaques arise in the brain at least 30 years after the fatty streaks of fetal arteries (Finch, 2005). The prevalence of dementia after age 80 ranges widely from 10% or lower to more than 50%, depending on the population (Fig. 1.10A). These indi- vidual outcomes in aging may derive from the same gene-environment interac- tions that influence vascular aging and pathology, discussed in later chapters. 1.6.1. Neuropathology of Alzheimer Disease Brain amyloid generates inflammation that interacts with neuronal regression. During ‘normal aging,’ nearly all humans accumulate some form of solid brain Aβ (Delaere et al, 1993; Mizutani and Shimada, 1992; Morris and Price, 2001), which is a focus of local inflammatory reactions. Senile plaques are extracellular aggregates of amyloids and inflammatory proteins, best known for the β-amyloid peptide ‘Aβ’ of 42 amino acids (Aβ1-42 ), which is proteolytically derived from the amyloid precursor protein (APP). However, the molar proportion of Aβ and the many plaque components is not known. Senile plaque fibrillar Aβ1-42 is stained by the dye Congo red (‘congophilic’). Aβ deposits are very heterogeneous in morphology and degree of fibrils, ranging from the classic senile plaques with fibrillar amyloid to diffuse amyloid deposits that are not congophilic. The classic Alzheimer ‘neuritic plaque’ has abnormal neurites along with congophilic Aß fibrils, reactive glia, and inflammatory proteins (Fig. 1.10B). The curving dendrites near plaques are predicted to have slowed neurotransmission (Knowles et al, 1999). Synaptic density (dendritic spines) drops sharply in neurons passing near neuritic plaques in transgenic mice (Spires et al, 2005). The inhibition of neurite outgrowth by aging glia (Fig. 1.9) may be a model for the glial contribution to abnormal neurites in senile plaques. Brain amyloid pools are very dynamic. Aβ is transported in both directions from the periphery across the blood-brain barrier. Slight increases in production of the Aβ peptide from extra gene copies (Down syndrome, transgenic mice) could enable slow plaque accumulation. Conversely, brain amyloid can be removed by circulat- ing antibodies in the transgenic mice and, possibly, in Alzheimer patients. Insulin and IGF-1 may also influence brain Aβ peptide pools (Section 1.6.5, below). Although amyloid accumulations are universal or nearly so during later aging, neuritic plaques are not found in all nondemented elderly (Braak and Braak, 1991; Price and Morris, 1999). Diffuse amyloid deposits are not fibrillar and have fewer inflammatory components (Braak and Braak, 1991; Price and Morris, 1999). Although diffuse plaques are not associated with cognitive deficits, nearby neurons show subtle changes (D’Amore et al, 2003). Various vascular abnormalities (microangiopathies) arise during aging and Alzheimer disease. Microvessels proliferate, with greater density and cork-screw- like tortuosity in later Alzheimer disease (Perlmutter et al, 1990). Angiogenesis is associated with senile plaques (Perlmutter et al, 1990; Wegiel et al, 2003) and in Inflammation and Oxidation in Aging and Chronic Diseases 87
  • the basilar arterioles of Alzheimer brains and transgenic mice (Beckmann et al, 2003; Burgermeister et al, 2000; Calhoun et al, 1999; Krucker et al, 2004; Van Dorpe et al, 2000). Plaques are next to, or penetrated by, one or more cerebral microvessels in human dementia (Ishii, 1958; Kawai et al, 1990; Wegiel et al, 2003). New plaques may arise from the budding of large plaques close to the perivascular zone (Wegiel et al, 2003). These findings suggest a role for the angiogenic inflammatory factors that increase in senile plaque genesis (see below). Vascular tortuosities during aging may be increased by hypertension (Akima et al, 1986; Hiroki et al, 2002; Moody et al, 1997). Transgenic Alzheimer mouse models also show a lower vascular density preceding the deposits of brain amyloid and vascular amyloid (Krucker et al, 2004). Blood flow slows, even before amyloid deposits (Niwa et al, 2002). We must also consider the 30–50% age-related decrease of microvasculature, shown in two rat strains (Sonntag et al, 1997; Sonntag et al, 2000), which may overlap with the vascular changes in transgenic Alzheimer mice. Growth hormone and IGF-1 contribute to these changes in aging rats (see below). Neurofibrillary tangles are the other Alzheimer hallmark as reported in Alzheimer’s original case of pre-senile dementia (Alzheimer, 1911). These intra- neuronal bodies are abnormally configured microtubule proteins (normally part of the cytoskeleton). Neurofibrillary tangles are paired filaments of polymerized hyperphosphorylated tau, an accessory microtubule protein, and do not contain the Aβ peptide. Besides their prominence in Alzheimer brains, tangles also accu- mulate sporadically during ‘normal’ aging absent Alzheimer disease (Mizutani and Shimada, 1992; Morris and Price, 2001). All nondemented centenarians examined had some tangled neurons (Silver et al, 2002). Of major importance to the evolution of human longevity (Chapter 6), aging great apes have very modest Aβ accumulations (Gearing et al, 1997), in contrast to their abundance in aging rhesus monkeys (Finch and Sapolsky, 1999; Finch and Stanford, 2004). Alzheimer-like changes also arise to some extent in other mammals. The accumulation of brain Aβ aggregates may be common in verte- brates during aging, because the Aβ peptide is highly conserved (Section 1.2.2, Fig. 6.2). Despite its importance for postmortem diagnosis, the amount of fibrillar Aβ is weakly correlated with the degree of neurodegeneration and cognitive deficits (Klein et al, 2001; Terry et al, 1991). Longitudinal clinical assessments by the Clinical Disease Rating (CDR) scale CDR 1-5 also recognize CDR 0.5 as a pre- clinical stage with subtle cognitive changes (Morris, 1999; Morris and Price, 2001). CDR 0.5 also overlaps with another classification of ‘mild cognitive impairment’ (MCI) (Petersen, 2004). CDR 0.5 brains show extensive neuron death, senile plaques, and neurofibrillary degeneration (Price and Morris, 1999; Price et al, 2001), corresponding to Braak neuropathology stages III to V (Fig. 1.17) (John Morris, pers. comm.). Cognitive dysfunctions correlate 4-fold better with synapse loss than amyloid load. Synapse loss represents both neuron loss and neuron atrophy (Section 1.2.2). 88 The Biology of Human Longevity
  • Inflammation and Oxidation in Aging and Chronic Diseases 89 0 40 30 20 10 0 40 30 20 10 0 40 30 20 10 0 40 30 20 10 0 40 30 20 10 0 40 30 20 10 Stage 0 Stage V - VI Stage IV Stage III Stage II Stage I 40 50 60 70 80 Y 3.5 0 0.5 1 1.5 2 2.5 3 Stage,mean 40 45 60 65 70 75 80 855550 Y A B FIGURE 1.17 For legend see page 90.
  • 90 The Biology of Human Longevity FIGURE 1.17 Age-increase in Braak stages of Alzheimer disease, based on the density of neurofibrillary tangles (NFT) in the temporal lobe, including the hip- pocampus. Postmortem brains (887 Ss, 20–104 y, 47% men and 53% women) from Mainz and Frankfurt University hospital autopsies. Exclusions included brain tumors, bleeding, and inflammation; prior cognitive status is unknown (Braak and Braak, 1991; Braak et al, 1998). (Redrawn from Ohm et al, 1995.) A. Average Braak Stage score by age. Stage 0, absence of NFT; Stages 1-2, NFT confined to entorhinal and transentorhinal cortex; Stages 3–4, spread to other limbic areas; Stages 5–6, exten- sive NFT at Khachaturian criteria (Khachaturian, 1985) for Alzheimer diagnosis. B. Distribution of Braak stages by age. Estimated times between stages: I to II, 16+ y; II to III, 14 y; III to IV, 13 y, IV to V, 5 y. The duration of Alzheimer pathogene- sis may be 50 y from the first NFT into end clinical stages. 9 Readers note my commercial interests in Alzheimer disease as a cofounder of Acumen Pharmaceuticals, Inc., with William Klein (Northwestern University) and Grant Krafft (Acumen). Excess Aβ in the brain is widely believed the major, if not the primary cause, of Alzheimer disease (Selkoe, 2000; Yankner, 2000). The gaps between evidence that Aβ initiates Alzheimer disease and the weaker correlation of cognitive changes with the amyloid load are known as the ‘amyloid conundrum’ (Klein et al, 2001). Strong evidence comes from overproduction of APP and Aβ. Down syndrome (tri- somy 21) gave important early support for the amyloid hypothesis because the trisomy 21, with its extra copy of the APP gene, increases Aβ production. By age 50, all Down brains have neuronal tangles, neuronal Aβ, and various extracellular Aβ including the classic amyloid plaques (Lott, 2005; Nixon, 2005; Sparks, 1996). In a rare case of partial trisomy of chromosome 21 that did not include the APP locus, this individual lived to age 78 without Alzheimer changes (Prasher et al, 1998). A mouse model of Down syndrome (trisomy chromosome 16) shows simi- lar effects from deleting the extra APP gene (Gardiner, 2005). Transgenic mice overexpressing normal or mutant human APP develop early onset deposits of Aβ. Although several transgenic AD mice have synaptic deficits and impaired memory, neurofibrillary degeneration and neuron death are lacking (Holcomb et al, 1998; Jankowsky et al, 2004; Jankowsky et al, 2005; Mucke et al, 2000). The full package of amyloid and neurofibrillary degeneration occurs in a triple transgenic mouse with mutant APP and tau (LaFerla and Oddo, 2005; Oddo et al, 2003a,b). Fibrillar Aβ1-42 was the initial candidate for neurotoxicity in Alzheimer disease research (Yankner et al, 1989; Yankner, 2000), while others argued that fibrillar Aβ1-42 is neuroprotective (Lee et al, 2005). However, Aβ1-42 oligomers show more neurotoxicity than the classical fibrils. A subclass of particularly toxic oligomers is designated ADDLs (amyloid-derived diffusible ligands).9 Oligomeric Aβ1-42 has
  • greater neurotoxicity than fibrillar Aβ (Klein et al, 2001; Oda et al, 1995) and is elevated >50-fold in Alzheimer brains (Gong et al, 2003). In rodent models, ADDLs impair memory and long-term potentiation (LTP) (Cleary et al, 2005; Klein et al, 2001; Lesne et al, 2006; Walsh et al, 2005), possibly by binding to synapses (Lacor et al, 2004). Given the adverse activities of the Aβ1-42 peptide, what might be its normal functions? The near invariance of Aβ1-42 sequence across vertebrates from fish to birds to primates implies some long-standing function. APP is axonally trans- ported to the presynaptic terminals (Buxbaum et al, 1998; Lazarov et al, 2005) by direct binding to the anterograde motor, kinesin (Kamal et al, 2000). Neuronal activity modulated Aβ secretion in the hippocampal slice model, which caused reversible synaptic depression (Kamenetz et al, 2003). Synaptically released Aβ appears to accumulate as plaques in transgenic mice (Lazarov et al, 2002; Sheng et al, 2002). Moreover, APP, Aβ, and the APP-like proteins (APPL1 and APPL2) may have roles in neuronal differentiation during devel- opment (Kimberly et al, 2005; Millet et al, 2005; Roncarati et al, 2002; Teo et al, 2005; Torroja et al, 1999). APP and Aβ may also participate in proinflammatory stress responses that overlap with the acute phase (Tuppo and Arias, 2005). Alzheimer transgenic mice given chronic LPS systemically (12 weekly i.p. injec- tions) had high neuronal levels of APP and Aβ (Sheng et al, 2003). These responses to LPS are consistent with the inflammatory processes in Alzheimer disease (see below) and the acceleration of Aβ deposits by HIV infections and LPS (Chapter 2). 1.6.2. Inflammation in Alzheimer Disease Senile plaques include many inflammatory proteins also found in atheromas (Table 1.3), some of which can stimulate free radical production, e.g., cytokines (IL-6, TNFα) and complement factors. C1q, the initiator of the classic complement cas- cade, is directly activated by Aβ aggregates (Fan and Tenner, 2004; Rogers et al, 1992; Webster et al, 2000). Further C-system enzymes yield the peptides C3a, C4a, and C5a; these ‘anaphylactic peptides’ can induce microglia/monocytes to produce free radicals. Anaphylactic peptides early in Alzheimer disease are indicated by the C3c and C4d fragments present in the pre-clinical stage CDR 0.5 (Zanjani et al, 2005). The classic senile plaques of advanced Alzheimer disease have numerous reactive microglia (macrophages). Microglia are of bone marrow origin and express CD68 and other monocyte-lineage epitopes also found in atheromas. However, in contrast to atheromas (see above), B- or T cells are rare in senile plaques. Reactive astrocytes around plaques may phagocytose Aβ (Thal et al, 2000; Wyss-Coray et al, 2003), whereas in Alzheimer transgenic mice, microglia near plaques did not (Stalder et al, 2001). Early in the modern era of Alzheimer research, it was suspected that brain inflammatory proteins were artifacts of blood-brain barrier breakdown before death. However, we (Johnson et al, 1992a,b; May et al, 1990; Oda et al, 1994; Inflammation and Oxidation in Aging and Chronic Diseases 91
  • Pasinetti et al, 1992) and others (Akiyama et al, 2000; Walker and McGeer, 1992) showed that C-system genes are expressed in normal brain astrocytes, microglia, and neurons, and are further induced during neurodegeneration. Many inflammatory proteins found in plaques also increase during aging in the brain (Table 1.5) in the absence of Alzheimer disease, as well as in other tis- sues (Section 1.8). Complement activation products C3c and C4d are found in diffuse Aβ deposits of clinically healthy elderly without indications of Alzheimer disease or gross neurodegeneration (Fig. 1.18) (Zanjani et al, 2005). Rodents also increase C1q and other inflammatory gene expression during brain aging (Pasinetti et al, 1999; Prolla and Mattson, 2001; Weindruch et al, 2002). Because rodent brains do not accumulate amyloid during aging, we can infer that in human brains there is a subset of inflammatory changes during normal aging that is independent of brain amyloid deposits. Corpora amylacea are another inflammatory aggregate accumulated during aging and further in Alzheimer brains (Cavanagh, 1999; Singhrao et al, 1995). In these microscopic extracellular bodies (3–15µ), complement proteins surround a polyglucosan core. Angiogenic-inflammatory factors also increase. VEGF and TGF-β1 are 5-fold higher in cerebrospinal fluid in both Alzheimer and vascular dementia (Tarkowski et al, 2002; Vagnucci and Li, 2003). As noted above, new microvessels arise near plaques. The interactions of oxidation and inflammation in amyloid plaques are less resolved than in atheromas. Plaques may represent feed-forward inflammatory processes. In 1982, Eikelenboom and Stam (1982) detected complement fac- tors in senile plaques and postulated roles in neurodegeneration. A few years later, IL-1 production was identified with microglia (Giulian et al, 1986). In 1989, Griffin and colleagues (Griffin et al, 1989) proposed that microglial activation and IL-1 induction early in Alzheimer stimulate excess production of the amy- loid precursor protein (APP) (Griffin, 2005). IL-1 also induces secretion of several complement factors by microglia (Veerhuis et al, 1999). Insulin and IGF-1 are also 92 The Biology of Human Longevity TABLE 1.5 Inflammatory Changes in Alzheimer Senile Plaques and Normal Aging Brain. Adapted from Finch (2005) Senile Plaque Aging Human Aging Rodent glial activation: GFAP, astrocytes; + + + + MHCII, microglia α1 -antichymotrypsin + α2 -macroglobulin apoE, apoJ, CRP, HOX-1, RAGE + + + +a Complement C1q, C3 + + corpora amylacea + C1q mRNA Cytokines: IL-1, IL-6, TNFα + + + + CRP is not expressed in rodent brains.
  • Inflammation and Oxidation in Aging and Chronic Diseases 93 FIGURE 1.18 Increased complement deposits (C3c, C3d, C4c, C4d) on diffuse amyloid Ab deposits in normal aging human brain (Clinical Disease Rating, CDR = 0). From collaboration with Joel Price and John Morris (Washington University) (Zanjani et al, 2005). implicated in amyloid deposition and clearance (see below). The hypothesis that Aβ overproduction is linked to inflammatory processes is being tested by drugs and diet. As discussed in Chapters 2 and 3, non-steroidal anti-inflam- matory drugs (NSAID ibuprofen) and diet restriction attenuate amyloid deposits in transgenic mice. Other proinflammatory cascades may be driven by glyco-oxidation (Section 1.2.6 and 1.4.2; Query II). Plaques accumulate glycated proteins and oxidized lipids in Alzheimer disease (Girones et al, 2004; Reddy et al, 2002; Wong et al, 2001) and in transgenic Alzheimer mice (Munch et al, 2003). AGE adducts (Smith et al, 1994) colocalized with iNOS (Wong et al, 2001), a source of free radicals (Fig. 1.11). In Alzheimer transgenic mice, intracellular AGE colocalized with IL-1β and TNFα in astrocytes associated with amyloid deposits (Munch et al, 2003). Glycated pro- teins can induce oxidative stress and inflammatory responses (Section 1.2.7). The glycation of Aβ increased microglial inflammatory responses (Gasic-Milenkovic et al, 2003). Neurofibrillary tangles also become glycated, with subsequent
  • generation of oxidative stress (Yan et al, 1995). Cause and effect are not resolved, because degenerating or stressed cells often attract macrophages and cause further inflammatory responses. Lastly, systemic inflammatory factors that increase the risk of vascular events (Fig. 1.16) were also associated with risk of cognitive decline and Alzheimer disease in two prospective studies. In the Honolulu-Asian Aging Study (Schmidt et al, 2002) and the Health, Aging, and Body Composition Study (Yaffe et al, 2004), serum CRP elevations were associated with subsequent dementia (Section 2.6, Fig. 2.8). Neuronal CRP also increases during Alzheimer disease (Yasojima et al, 2000). There may be links of inflammatory processes of aging to the accelerating incidence of Alzheimer disease (Section 2.7). 1.6.3. Prodromal Stages of Alzheimer Disease The onset of Alzheimer neurodegeneration is not as well defined as for vascu- lar disease. The most detailed analysis comes from the Heiko and Eva Braak’s huge autopsy series (Fig. 1.17). Neurofibrillary tangles, neuritic plaques, and various other Aβ deposits accumulate to some degree in almost all brains during aging (Fig. 1.10B). The six Braak stages, however, are based only on the neurofibrillary load (Fig. 1.17). Tangles (and plaques) appear to spread from subregions of the frontal cortex to the underlying hippocampus. Stage 0 rep- resents brains without any tangles. Stage I, with a few localized tangles, is rare before 40 y. Later Braque stages increase after 65 y, consistent with other cognitive assessments and association with neuropathology (Kawas and Katzman, 1999; Khachaturian, 1985). Stage VI is the end stage of senile dementia. This prolonged sequence may span 50 years from initial neurofibrillary changes until definitive dementia (Ohm et al, 1995). Neuronal endosomes show early (prodromal) changes. Endosomes mediate vesicle recycling and process the amyloid precursor protein (APP) (Nixon, 2005). Preclinical stages (Braak stages I and II of elderly brains) had enlarged endo- somes and higher soluble Aβ, but no extracellular fibrillar amyloid (Cataldo et al, 2004). Late stages have extensive endosomal enlargement in neurons (up to 30- fold) (Nixon, 2005). Nixon and colleagues hypothesize that neuronal endocytic abnormalities are an early step in pathogenesis that increases the production of soluble Aβ. Other evidence comes from Down syndrome. Preceding the univer- sal, early adult onset Alzheimer pathology, fetal Down brains have enlarged neu- ronal endosomes, with further deposition of extracellular Aβ deposits (Cataldo et al, 1997; Nixon, 2005). In intraneuronal Aβ1-43 was detected in young Downs (neonate to 28 y), which had no extracellular Aβ (Hirayama et al, 2003). A tri- somic mouse model of Downs also showed early endosomal pathology (Galdzicki and Siarey, 2003; Nixon, 2005). ApoE alleles influence these changes (details in Section 5.7). The apoE4 allele, relative to apoE3, accelerates neurodegeneration in familial Alzheimer disease and in Down syndrome (Del Bo et al, 1997; Isacson et al, 2002). ApoE4 has clear 94 The Biology of Human Longevity
  • effects before age 50. In Braak Stage I brains aged 22–46 y, apoE4 was 2-fold overrepresented relative to non-carriers (Ghebremedhin et al, 1998). Moreover, apoE allele associations are linked to combinations of eight other inflammatory genes in Alzheimer risk (Licastro et al, 2006). Functional effects emerge early in E4 carriers. By PET imaging, asymptomatic E4 carriers have lower cerebral glucose metabolism in the frontal cortex even in their 30s (Reiman et al, 2004; Small et al, 2000). Further decreases of metabolism ensue at clinical stages (Alexander et al, 2002; Small et al, 2000). These metabolic impairments imply cell changes decades before clinical disease, consistent with the 30-year duration of Braak stages II to IV. Other cognitive declines are also influ- enced by apoE4. ApoE4 also predicted faster cognitive decline in the MacArthur Studies in Successful Aging (Bretsky et al, 2003). However, associations of later cog- nitive loss with midlife hypertension with apoE4 in the Honolulu Asia Aging Study (HAAS) (Peila et al, 2001) were not found by other studies (Qiu and Fratiglioni, 2005). A new concern is that apoE4 may influence brain development. In mice car- rying human apoE genes, cortical neurons have less dendritic complexity in huE4/E4 mice than in huE3/E3 mice (Wang et al, 2005a). 1.6.4. Overlap of Alzheimer and Cerebrovascular Changes Readers may have noticed a deviation from convention. Designating these senile brain diseases collectively as ‘Alzheimer disease and vascular-related dementias’ diverges from the convention that carefully discriminates these conditions. Alzheimer plaques and tangles and cerebrovascular lesions often co-occur in the elderly demented. Alzheimer disease and vascular dementia share many of the same risk factors (Chapter 3) and may benefit from many of the same diets and drugs (Chapter 2). This evidence, taken with the inflammatory proteins of senile plaques, suggests that Alzheimer and vascular dementia share key inflammatory processes. Alzheimer plaques and tangles can arise in the absence of cerebrovascular lesions (Kemper, 1984; Kidd, 1964; Wisniewski and Terry, 1973). These distinc- tions were established in 1970 by the pioneering Newcastle Study (U.K.) (Tomlinson et al, 1968; Tomlinson et al, 1970). The conclusion that cerebrovascu- lar and Alzheimer pathology can arise independently during aging remains valid today (Chui, 2005; Reed et al, 2004a; Terry et al, 1999). However, the combination of Alzheimer pathology and microinfarcts is commonly observed in dementia cases (Tomlinson et al, 1970; Petrovich et al, 2002). The traditional term cerebrovascular disease now includes multiple types of changes in the cerebral vasculature that are hard to summarize because of their diversity: These range from focal infarcts due to a single thrombosis that causes local neuron death to more distributed hyaline degeneration of arterioles (arterioloscle- rosis) that remain patent but cause chronic hypoperfusion. Helena Chui suggests the broader term cerebrovascular-related brain injury (Chui, 2005). The best rec- ognized lesions of cerebrovascular aging are infarcts (Jagust, 2001; Langa et al, 2004; Inflammation and Oxidation in Aging and Chronic Diseases 95
  • Reed et al, 2004a; Roman et al, 2004). Infarcts may be additive with the density Alzheimer plaques and tangles in predicting the level of dementia (Fig. 1.19) (Schneider et al, 2004). Depending on their location, infarcts may not cause cognitive impairments. White matter damage is also associated with vascular lesions and may contribute to dementia by slowing the neuronal conduction, which is crucial for high-speed transmission between cortical centers. Glial inflammatory changes of aging are implicated (see below). Current cognitive tests do not resolve vascular versus pure Alzheimer con- tributions to dementia (Chui, 2005; Reed et al, 2004a). First, all studies so far are samples of convenience or of special groups that may not represent pop- ulations. Second, no ‘simple metric’ identifies vascular dementia. While large infarcts (>50 mL volume) are strongly associated with dementia, small infarcts in key locations also cause dementia. Third, no study has fully characterized premortem cerebral metabolism with postmortem cerebrovascular local blood flow, cerebrovascular amyloid, and both macroscopic and microscopic 96 The Biology of Human Longevity 1 0.5 0 0 1 2 3 Alzheimer Pathology ProbabilityofDementia Infarcts No infarcts FIGURE 1.19 Clinical dementia risk increases with cerebrovascular damage and the level of Alzheimer disease characteristic neuropathology in early stages of AD. The probability of dementia diagnosis before death versus AD pathology with cerebral infarction (solid line) and without infarctions (dashed line). From The Religious Orders Study (both sexes; mean age, 84 y; 153 Ss; 44% were demented; postmortem, 99% of brains had NFT; 79%, neuritic plaques, 84%, diffuse amyloid; 35%, macroscopic infarcts; 29%, microscopic infarcts.) Macroscopic infarcts con- tributed progressively less to dementia with higher levels of AD neuropathology. (Text and figure adapted from Schneider et al, 2004.)
  • infarcts. Vascular amyloid Aβ also accumulates (‘congophilic angiopathy’) in association with white matter damage (Kalback et al, 2004) and increased risk of aneurysms (Van Dorpe et al, 2000). It is not known how vascular amyloid or aneurysms are related to ‘prodromal’ lipid accumulations in cerebral arter- ies (Fig. 1.15B), or to arterial ‘fibrohyaline thickening,’ which typically emerges by age 40 (Section 1.5.1). The vascular contribution to dementia may be stronger in the future as longevity continues to increase (Roman et al, 2002). The boundaries between ‘usual aging’ and Alzheimer disease are less clear at later ages. Fourth, populations differ in the proportions of dementias. Vascular dementia may be 50% more prevalent in Japan than in U.S. Caucasians (Fujishima and Kiyohara, 2002; Jorm, 1987). Hawaiian Japanese who adopted Western diets may have less vascular dementia than on ‘oriental’ diets (Ross, 1999). However, a prospective study did not find associations of blood cholesterol with Alzheimer risk; as expected, apoE4 increased Alzheimer risk (Li et al, 2005). Two African- American samples had up to 2-fold more dementia than Caucasians (Demirovic et al, 2003; Mayeux, 2003a,b). Moreover, Nigerian Yorubans in Ibadan showed 50% less dementia than African Americans in Indianapolis (Hendrie et al, 2001). Indian elderly may also have less dementia (Indo-U.S. Study) (Chandra et al, 2001). These ethnodemographic differences in dementia have not been exam- ined for neuropathology. A precedent is population differences in cancer. Breast cancer in Japan in the 1980s was 50% less at all ages than in Japanese born in the United States or U.S. Caucasians; these differences have since decreased in association with increased body fat (Pike et al, 1983; Probst-Hensch et al, 2000; Ursin et al, 1994). Changes in cerebral blood flow and metabolism during aging may overlap with dementia. As noted above, ApoE4 carriers who are asymptomatic during middle age already have slightly depressed frontal cortex metabolism (Reiman et al, 2004; Small et al, 2000). Older patients with dementia or asymptomatic atherosclerosis typically have 10% or greater reductions in blood flow than healthy elderly (Meyer and Shaw, 1984; Schaller et al, 2005). However, cerebral blood flow also declines during normal aging by 15% from 30 to 65 y (Amano et al, 1982; Melamed et al, 1980; Plaschke, 2005) (Fig. 1.20A). These observations on healthy subjects without risk factors for vascular pathology confirm the pioneering observations of Seymour Kety (Kety, 1956), which were initially discounted because of presumed confounds by asympto- matic atherosclerosis (Meyer and Shaw, 1984). Moreover, aging rats have progressive decrease in arteriolar density to about 35% across the adult life span, with similar changes in cerebral blood flow (Sonntag et al, 1997; Sonntag et al, 2000) (Fig. 1.20B legend). These striking changes hold for two rat genotypes that do not show occlusive cerebral atherosclerosis and are consistent with the cerebral blood flow decrease in normal human aging. It is time to revisit Kety’s hypothesis that the age decline in blood flow is due to reduced cerebral metabolism (Kety, 1956) (see above). Other studies show Inflammation and Oxidation in Aging and Chronic Diseases 97
  • 98 The Biology of Human Longevity FIGURE 1.20 Brain vasculature and aging. A. Cerebral cortex gray matter blood flow declines pro- gressively during normal human aging by 25%, 20–80 y, or about 0.4%/y (my calculation). (Redrawn from Amano et al, 1982). Cerebral blood flow (BF) was measured by the stable xenon flow CT tech- nique on 13 healthy volunteers without vascular risk factors or diabetes, and normal cognition on the WAIS; the mean and range are shown for each subject; r of means = 0.88 (P<0.001). Cortical white matter also declined in BF with a shallower age gradient, but the age gradients were similar in gray matter cortex, basal ganglia, and thalamus. These data confirm (Melamed et al, 1980) and (Kety, 1956). B. Cerebral vasculature loss in aging rat. Cortical surface arterioles and venules in 13- and 29-m-old Brown Norway (BN) male rats. The progressive decrease in arterioles per surface area rela- tive to age 5 m to 13 m, 1–5%; and 5 m to 29 m, −40%. Arteriolar anastamoses also decreased, by ca. 10%. Cerebral blood flow changed about 30% over the life span, close to human aging changes. The F344/BN hybrid changed similarly. Growth hormone injections for 35 d partly reversed the arte- riolar loss in rats aged 30 m, but did not alter the young. (From Sonntag et al, 1997.) 100 80 60 40 20 10 LCBF(ml/100gbrain/min) Age (YR) 20 A 40 60 80 y = 95.9 - 0.37x (r = 0.88, P < 0.001) 5 10 15 1 2 3 4 10 20 30 Young AL Old AL Old DR Young AL Old AL Old DR Young AL Old AL Old DR #/mm2 #/mm2 #/mm2Arteriolar Density Anastomotic Density Venous Density Old ALYoung AL Old DR B * * *
  • Inflammation and Oxidation in Aging and Chronic Diseases 99 more or less concurrent synaptic atrophy in normal humans and rodents during middle age (Masliah et al, 1993; Morgan et al, 1987; Morgan et al, 1990; Rozovsky et al, 2005). Moreover, apoE4 carriers show reduced cerebral metabolism during middle age, as noted above (Reiman et al, 2004) (Section 5.7.4). We are evaluat- ing whether glial activation from inflammation and/or oxidative stress is a pri- mary factor in synaptic atrophy, which secondarily causes the reduction of cerebral blood flow (Fig. 1.7E). Blood pressure is another shared risk factor with vascular events and demen- tia. The large ARIC Study and eight others found that middle age hypertensions increased risk of later cognitive declines (Qiu et al, 2005). Vascular tortuosities during aging may be increased by hypertension (Akima et al, 1986; Hiroki et al, 2002; Moody et al, 1997). Antihypertensive drugs may decrease the risk of vas- cular and Alzheimer dementia (Qiu et al, 2005). Possible pathways include hypoperfusion from cerebral atherosclerosis. In rodent models, chronic hypop- erfusion caused large-scale activation of microglia, as in Alzheimer brains (Farkas et al, 2004). Obesity as a risk factor is discussed below. 1.6.5. Insulin and IGF-1 in Vascular Disease and Alzheimer Disease The inter-relations of obesity, diabetes, and glucose dysregulation are among the huge frontiers in medicine with great importance to vascular disease and possi- bly also to Alzheimer disease independent of the cerebrovasculature. The meta- bolic hormones insulin and IGF-1 are increasingly implicated in atherosclerosis and Alzheimer disease and could be critical links to the longevity pathways iden- tified in mutant mice, flies, worms, and yeast (Fig. 1.3A, B) (Chapter 5). However, IGF-1 deficiencies show opposite effects by increasing the risk of cardiovascular disease and congestive heart failure in clinical studies and experimental models. These hormonal effects of insulin pathways are relatively new mechanisms and were first considered about 1985 for vascular disease in diabetics (Koschinsky et al, 1985) and a decade later for Alzheimer disease (Finch and Cohen, 1997). Low serum IGF-1 is associated with vascular disease (Conti et al, 2004). Elderly in the Framingham Heart Study with the lowest IGF (serum IGF-1 <75 µg/L; lowest decile) had a 2.6-fold higher risk of congestive heart failure; during a 5-year follow-up, the hazard decreased by 27% for every standard deviation increment (Vasan et al, 2003). Low serum IGF-1 may be an independent risk factor, because the associations with vascular event did not depend on choles- terol, age, and other risk factors (Conti et al, 2004), nor on serum IL-6, which is commonly elevated in elderly and in vascular disease and that can lower IGF-1 (De Benedetti et al, 2002). Another association is with arterial plaque instability, which may be increased by low IGF-1 and low PAPPA-A, a protease which cleaves IGF-1 binding protein to release IGF-1 (Beaudeux et al, 2003). Insulin/IGF-1 signaling is implicated in many aspects of vascular biology (Bayes-Genis et al, 2000; Frost et al, 1997; Gonzalez et al, 2001). Figure 1.3B shows the basic machinery in processes that can be cardioprotective or
  • proatherogenic (Conti et al, 2004). Activation of IGF-1 receptors, in turn, activates PI3-kinases, serine/threonine kinase Akt, which then phosphorylates the consti- tutive NOS (nitric acid synthase) in the vascular endothelia. The increased nitric oxide (NO) production has multiple cardioprotective actions, which include vasodilation, platelet inhibition, and free radical scavenging. Angiotensin II inhibits this pathway, while TNFα, which is increased in atherosclerotic plaques, can inhibit IGF-1 expression (Anwar et al, 2002). IGF-1 may also be pro-athero- genic by stimulating the proliferation of vascular smooth muscle cells. Attempts to pharmacologically manipulate IGF-1, e.g., by manipulating growth hormone (GH) secretion with the somatostatin analogue angiopeptin, have not been effec- tive (Conti et al, 2004). Leptin signaling may also converge with insulin/IGF-1 pathways in regulating cell proliferation (Tapia, 2005). Leptin in obese patients shows modest correlation with telomere loss (Valdes et al, 2005), which will be considered further below in relation to fat as a source of inflammatory factors. The decline of IGF-1 and GH with normal aging is receiving attention as a major cause of insulin resistance during aging. GH secretion by the pituitary gradually declines during aging at the rate of 10–15%/decade of adult life (Conti et al, 2004; Obermayr et al, 2005; Rudman et al, 1981). GH is a major regulator of IGF-1 expression and secretion by liver, kidney, and vascular smooth muscle cells. Hepatic secretion may account for the bulk of serum IGF-1 decline. Age changes in GH and IGF-1 were partly reversed by donepezil, a cholinesterase inhibitor (Obermayr et al, 2005). Healthy centenarians may have higher IGF-1 (Paolisso et al, 1997; Paolisso et al, 1999; Franceschi et al, 2005) (Fig. 5.11). Insulin/IGF-1 functions are also implicated in brain vascular aging (Sonntag et al, 1997; Sonntag et al, 2000). As noted above, rat brain shows striking decrease of vascular density. These major declines were partly reversed by injec- tions of GH for 35 d, which increased plasma IGF-1 (Fig. 1.20 legend). Cerebral blood flow was also restored. Although these striking age changes and responses to GH have not led to clinical trials, there is reason to consider that the reduced cerebral blood flow during ‘normal’ aging (see above) may be also linked to the decrease of GH and IGF-1. Another emerging possibility is the role of IGF-1/insulin in Alzheimer dis- ease from epidemiological and clinical associations with hyperinsulinemia and maturity-onset diabetes (Carro and Torres-Aleman, 2004; Craft and Watson, 2004; Finch and Cohen, 1997; Gasparini and Xu, 2003; Steen et al, 2005). Blood insulin and IGF-1 are actively transported across the blood-brain barrier, which expresses relevant receptors (Steen et al, 2005). The importance of insulin transport was demonstrated by a recent study with healthy volunteers (Fishel et al, 2005). Induced hyperinsulinemia rapidly increased cerebrospinal fluid (csf) cytokines (IL-1α, β, IL-6, TNFα), Aβ1-42 , and F2-isoprostane, a marker of oxidative stress. Serum and csf cytokines were uncorrelated, implying that the insulin effects are not due to peripheral transport. However, plasma Aβ1-42 was increased in association with elevations of csf transthyretin, a transporter for Aβ1-42 from the 100 The Biology of Human Longevity
  • CNS to the peripheral blood. In a mouse model, serum IGF-1 also enhanced Aβ transport from the brain to peripheral blood by the amyloidogenic transthyretin (Carro and Torres-Aleman, 2004). Plasma IGF may be lower in sporadic and familial Alzheimer disease (Swedish APP670/671) (Mustafa et al, 1999). Within the brain, the degradation of Aβ1-42 may compete with insulin for IDE (insulin-degrading enzyme), which is also a candidate gene for Alzheimer dis- ease (Gasparini and Xu, 2003; Farris et al, 2004; Leissring et al 2003; Bertram et al, 2007). Moreover, IGF-1 can be neuroprotective against Aβ (Aguado-Llera et al, 2005; Dore and Quirion, 1997) via Akt kinase (Zheng et al, 2000). Both insulin and IGF-1 and their receptors may be synthesized by brain neurons (Steen et al, 2005). Despite the major gaps, the present evidence strongly connects brain aging to the vascular and longevity pathways in insulin/IGF signal- ing. These metabolic associations are supported by dietary influences on Alzheimer disease (Chapter 3). The genetics of these relationships is discussed in Chapter 5. 1.6.6. Blood Inflammatory Proteins: Biomarkers for Disease or Aging, or Both? Elevated acute phase proteins imply current or future health impairments. Serum CRP and IL-6 increases on the average after middle age in most populations (Cesari et al, 2004; Ershler and Keller, 2000; Ferrucci et al, 2005; Wilson et al, 2002). For example, a large random sample in Tuscany, Italy (InCHIANTI) showed progressive increases of CRP, fibrinogen, and IL-6 (Fig. 1.21) (Cesari et al, 2004). No age changes were seen in IL-1, TNF-, and TGF-β1. Cardiovascular disease is a major factor in these increases. Age alone, separated statistically from disease, predicted increased CRP and IL-18 in women only. Other studies also show uneven distributions of acute phase proteins, possibly subpopulations with elevated vascular risk factors including obesity (see below) or periodontal dis- ease (Bruunsgaard et al, 1999a; D’Aiuto et al, 2005; Deliargyris et al, 2004) (Section 2.3.1). Old age IL-6 elevations strongly associate with disability and mortality (Ferrucci et al, 1999; Harris et al, 1999; Ishihara and Hirano, 2002; McCarty, 1997). High CRP and IL-6 doubled the mortality risk in the healthy, non-disabled of the Iowa 65+ Rural Health Study (Harris et al, 1999) (Fig. 1.22). InCHIANTI associated high CRP and IL-6 with poor physical condition and hand-grip strength (Cesari et al, 2004), while the MacArthur Study of Successful Aging associated high CRP with reduced recreational (voluntary) activity and social integration (Loucks et al, 2006). Genetic influences, while expected, have not been found. In the Women’s Health and Aging cohorts, IL-6 variants did not associate with plasma levels of serum IL-6 or frailty (Reuben et al, 2003). Again, no association was found between IL-1, IL-6, and TNF alleles and mortality in Finnish nonagenarians (Wang et al, 2001). Larger population samples may be needed (Section 1.4.3). Inflammation and Oxidation in Aging and Chronic Diseases 101
  • 102 The Biology of Human Longevity 1.5 1 0.5 0 −0.5 −1 −1.5 1.5 1 0.5 0 −0.5 −1 −1.5 1.5 1 0.5 0 −0.5 −1 −1.5 1.5 1 0.5 0 −0.5 −1 −1.5 20-39 40-49 50-64 65-74 75-84 85+ Y20-39 40-49 50-64 65-74 75-84 85+ Y Z score Z score IL-6 IL-6r IL-18 IL-6 IL-6r IL-18 CRP Fibrinogen CRP Fibrinogen Men Men Women Women FIGURE 1.21 Blood acute phase proteins and aging graphed as z-plots in units of standard deviation (SD) from the mean. InCHIANTI Study, 1998 sample; random sample from Tuscany, Italy populations; 30 men and 30 women from each decade, 20–69 y. (Redrawn from Ferrucci et al, 2005.) A provisional conclusion is that the average increases of CRP and IL-6 at later ages represent the increase of clinical sub- groups with various diseases and conditions that elevated acute phase responses. It is interesting to compare the age trends of blood proteins with the acute phase response in young adults, which are similar, with the possible exception of IGF-1 (Table 1.5). An open question is the role of infections (Chapter 2).
  • Inflammation and Oxidation in Aging and Chronic Diseases 103 1.7. INFLAMMATION IN OBESITY Obesity is associated with chronic low-grade inflammation that synergizes to increase the risk of Alzheimer disease, vascular events, and other adverse out- comes of aging, according to the body mass index (BMI),10 which is widely used to estimate obesity. For example, Alzheimer risk varied progressively with BMI in an 18-year prospective study (Gustafson et al, 2003): For each BMI unit increase at age 70 y, Alzheimer risk in women was increased by 36%; men, however, did not show these associations. There may be a female bias in mid-life obesity and later Alzheimer risk, of 2-fold in one study (Tabet, 2005). Those younger than 76 y showed a ‘U-shaped’ relationship between BMI and dementia, whereas at later ages, low BMI from weight loss may ‘mask’ these associations (Luchsinger et al, 2006). Mid-life obesity in combi- nation with high cholesterol and systolic pressure increased Alzheimer risk additively by 6.2-fold (Kivipelto et al, 2005). 10 The body mass index (BMI) is a crude but widely used measure of body fat, calculated as: height (m)/mass2 (kg). Underweight is BMI <18.5; normal weight, 18.5–24.9; over- weight, 25–29.9; obesity >30. The height-weight curves differ for men and women, and for children and adolescents. The BMI does not discriminate the type or location of fat, e.g., abdominal fat has a different physiology and pathology than subcutaneous fat (Chapter 3). 5.0 4.0 3.0 2.0 1.0 0.0 Low CRP & IL-6 High CRP High IL-6 High CRP & IL-6 Mortalityrisk FIGURE 1.22 High levels of blood IL-6 and CRP double mortality risk in healthy, nondisabled elderly. From the Iowa 65+ Rural Health Study (1,293 Ss followed 4.6 y). High: CRP 2.78 mg/L, IL-6 >3.19 pg/mL. (Redrawn from Harris et al, 1999.)
  • 104 The Biology of Human Longevity In current thinking about these complex pathologies, obesity is considered as a component of the ‘Metabolic Syndrome’ (Table 1.6), which is defined by three or more of the five traits that increase the risk of diabetes, cardiovascular disease, and chronic kidney disease. These traits overlap with ‘syndrome X’ or the ‘insulin- resistance syndrome,’ a cluster of metabolic dysfunctions (Kim et al, 2004; Reisin and Alpert, 2005; Reynolds and He, 2005). In some current populations, nearly 50% of adults meet ‘syndrome’ criteria. In a major prospective study of elderly, the com- bination of elevated CRP and IL-6 together with the ‘Syndrome’ increased the risk of cognitive impairment by 1.66-fold during the next 4.5 y (community living, 70–79 y; Health, Aging, and Body Composition Study) (Yaffe et al, 2004). In NHANES II, those with the ‘Syndrome’ had 2-fold higher mortality risk from coro- nary heart disease during 13 years of follow-up. Among those with both the ‘Metabolic Syndrome’ and coronary disease at entry, mortality was 4-fold higher (Malik et al, 2004). Chronic kidney disease risk is increased up to 5-fold in propor- tion to the number of ‘Syndrome’ criteria. The relationship of obesity to inflammation is fundamental because adipocytes release IL-6, TNFα, and other proinflammatory cytokines (Hukshorn et al, 2004; Lee et al, 2005; Toni et al, 2004). Blood inflammatory cytokines tend to increase with obesity (Table 1.7). There is also a striking correlation between blood CRP ele- vations and leptin (Fig. 1.23), which is secreted by adipocytes; CRP induction in hepatocytes may be mediated by IL-6 or TNFα (Shamsuzzaman et al, 2004). Abdominal visceral fat is correlated with plasma OxLDL and CRP (Couillard et al, 2005). Lipid peroxides are elevated 2-fold in obese humans (plasma, MDA assay) (Yesilbursa et al, 2005) and obese rats (urinary isoprostanes) (Dobrian et al, 2004). Leptin, besides regulating food intake (Chapter 3), is proinflammatory by enhanc- ing phagocytosis and inducing cytokines (Section 1.3.1). The inflammatory profile of obesity implies increased oxidative stress. Free rad- ical production (ROS) is increased in adipocytes of obese mice (Furukawa et al, 2004). Among many examples, the Framingham Study correlated BMI with plasma F2-isoprostanes, which are directly linked to lipid oxidation (8-epi-PGF2α , a stable by-product of arachadonic oxidation) (Keaney et al, 2003). TABLE 1.6 The Metabolic Syndrome Women Men waist circumference > 88 cm 102 cm serum triglycerides ≥ 150 mg/dL (1.7 mM) Same HDL-cholesterol < 50 mg/dL (1.3 mM) < 40 mg/DL (<1.03 mM) fasting blood glucose ≥ 110 mg/dl (6.2mM) Same blood pressure (systolic/diastolic) >130/85 mg Hg Same National Cholesterol Education Program’s Adult Treatment Panel (Anonymous, 2001). The choice 3 of 5 traits represents the uncertainties of definition. Gender and ethnicity also modify these five criteria. The clinical community broadly recognizes their value in describing the epidemic of obesity and diabetes.
  • Obesity also intensifies osteoarthritis and accelerates its onset (Bray and Bellanger, 2006; Rubenstein, 2005). Osteoarthritis is an age-related inflammation caused by mechanical stress, rather than by simple 'wear-and-tear’ as once thought. Osteoarthritis may be unavoidable during aging and is more intense in joints that bear the most weight. Osteoarthritis has traditionally been considered ‘non-inflammatory,’ in contrast to the severe inflammation of rheumatoid arthri- tis. Mechanical pressures that are increased by obesity activate catabolic responses of the articular chondrocytes to cause matrix loss and accumulation of AGEs (Loeser, 2004). Acute phase proteins (CRP, IL-1) and other inflammatory molecular responses arise early in osteoarthritis (Benito et al, 2005; Bonnet and Walsh, 2005; Igarashi et al, 2004; Loeser et al, 2004). Pain in osteoarthritis is linked to inflammation. The grinding of adjacent bones as cartilage erodes often causes pain. Other pain may come from osteophytes, spurs of new bone formed in arthritic joints, which press upon nerves; their genesis appears to depend on synovial macrophages (Blom et al, 2004). Anti-inflammatory drugs may reduce pain by inhibiting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), which produce prostaglandins and leukotrienes (Bonnet and Walsh, 2005; Hinz and Brune, 2004) (Chapter 2.9.3). Inflammation and Oxidation in Aging and Chronic Diseases 105 TABLE 1.7 Inflammatory Changes in Alzheimer Senile Plaques and Normal Aging Brain Acute Phase Responses and Aging Trends (bold shows differences) Acute Phase in Adults Normal Aging Trends Diabetes-Obesity blood amyloids serum amyloid A modest-large increase CRP blood complement C3 modest-large increase blood cytokines: CRP modest-large increase modest increase modest increase IL-1β IL-6 TNFα blood coagulation (fibrinogen) modest-large increase modest blood lipids HDL decrease decrease decrease LDL increase increase increase oxLDL increase increase increase triglycerides increase increase increase Cortisol modest-large increase modest increase modest increase Insulin insulin resistance resistance IGF-1 decrease increase Acute phase in adults (Wright et al, 2000). IGF-1 in aging, Fig. 5.11 and (Bonafe et al, 2003).
  • 106 The Biology of Human Longevity Lastly, I briefly mention that obesity is associated with modestly higher risks of some cancers, e.g., 10–20% higher risk of pancreatic cancer (Patel et al., 2005b; Berrington de Gonzalez et al, 2003) and advanced prostate cancer (Macinnis and English, 2006). Many factors are involved besides metabolic hormones or acute phase proteins. For example, the higher levels of endometrial cancer in post- menopausal obesity are associated with the production of estrone by fat depots, which drive endometrial cell proliferation. But again, exercise is a counterbalanc- ing factor, possibly through improved insulin sensitivity (Kaaks et al, 2002) (Section 3.4). 1.8. PROCESSES OF NORMAL AGING IN THE ABSENCE OF SPECIFIC DISEASES The shared inflammatory processes in the major chronic diseases discussed above also arise in tissues in the absence of these specific diseases. We have seen this already in arterial changes (Table 1.4). Some of the following exam- ples address Query I: that bystander damage from oxidative stress stimulates chronic inflammation. 0.0 0.5 1.0 1.5 2.0 −2 −1 0 1 LogCRP Log Leptin Normal Weight, BMI < 25 0.0 0.5 1.0 1.5 2.0 −2 −1 0 1 LogCRP Log Leptin Overweight and Obese, BMI ≥ 25 FIGURE 1.23 Blood C-reactive protein (CRP) and leptin correlations, graphed separately for BMI < 25 and > 25 (see footnote 10 for definition of BMI). CRP is elevations are strongly correlated with blood leptin, secreted by adipocytes; CRP may be regulated by IL-6 or TNFα, also secreted by adipocytes. (Redrawn from Shamsuzzaman et al, 2004.)
  • Inflammation and Oxidation in Aging and Chronic Diseases 107 1.8.1. Brain Microglia (macrophage/monocytic cells) show definitive activation during normal aging in rodent, monkey, and human. Overall, the increase of activated microglia during aging is 25–75% less than in Alzheimer brains (Finch et al, 2002). In rat cerebral cortex (Vaughan and Peters, 1974) and spinal cord (Stuesse et al, 2000), microglial activation progresses from maturity into old age (Fig. 1.8A) and is associated with increased production of IL-6 and TNFα (Xie et al, 2003). Humans have similar increases in activated microglia in the hippocampus during normal aging without neuropathology (DiPatre and Gelman, 1997). These cell changes are consistent with MRI imaging findings showing that white matter dis- organization begins during middle age (Fig. 1.8B). White matter inflammatory changes may be important to the usual slowing of information processing. Many of the same inflammatory proteins found in senile plaques also increase modestly in the brains of many mammals during normal aging in the absence of Alzheimer, e.g., cytokines and complement factors (Table 1.5). Complement proteins are found on the diffuse amyloid deposits of normal aging (Zanjani et al, 2005) (Fig. 1.18) and in corpora amylacea, discussed pre- viously regarding Alzheimer disease, which accumulate modestly in non- pathological brain aging (Singhrao et al, 1995). Because aging rodent brains lack amyloid Aβ, yet show comparable microglial activation to that in humans (see above), it may concluded that microglial activation during aging can be initiated independently of amyloid deposits. This is a major point in inter- preting the human aging changes, because tissue amyloids can activate microglia. For example, brain amyloid deposits increased the influx of blood- born monocytes, which are precursor cells of microglia (Fiala et al, 1998). Similarly, in hemodialysis patients, intestinal amyloid formed from β2- microglobulin are associated with activated macrophages (Miyata et al, 1994), which is a completely distinct protein from brain Aβ. 1.8.2. Generalized Inflammatory Changes in Normal Tissue Aging Other tissues besides the brain show increased inflammation during aging. The variability of tissue proteases (cathepsins) between studies (Finch, 1972a) is a clue to the importance of inflammation in aging: Because these enzymes are associated with macrophages, the variability between studies could reflect varia- tions of infiltrating macrophages, which are prominent in liver histology from aging rats in one early colony (Andrew et al, 1943). Data from rodent colonies before 1970 may be confounded by infections that have dwindled (Section 2.2.2). Nonetheless, in modern specific-pathogen free (SPF) colonies of aging rodents, lymphocytic infiltration is common and not considered pathological (Turturro et al, 2002). Fibrosis is also common in aging myocardium (Schriner et al, 2005) (Section 2.2) and other visceral organs. Gene expression profiling by microarray chips has given further examples of inflammatory processes during aging. Microarray profiling consistently increases
  • in many tissues during normal aging. Table 1.8 summarizes the consistent increase of inflammatory gene expression during aging in brain regions, liver, heart, and skeletal muscle. Inflammation-related genes may be the single largest category of change during aging (Table 1.9). Several complement (C) factor mRNAs increase in brain, liver, and myocardium of aging rodents. C1q is the initiating complex of the classical C-cascade. We showed the increase of C1q in the brain during normal aging, in the striatum (Pasinetti et al, 1999), as confirmed by microarray (Table 1.8). C-system genes also have greater expression in aging human frontal cortex (Erraji-Benchekroun et al, 2005; Lu et al, 2004; Pavlidis et al, 2004). Increased expression of the com- plement system during aging may contribute to C-factors in the corpora amylacea and in the deposits of C3 and C4 on diffuse brain amyloid in nonpathological cases (Fig. 1.18). The ‘anaphylactic peptides’ (C3a, C4a, C5a) can stimulate macrophage-monocyte cells in brain and elsewhere to produce ROS that cause oxidative damage. IL-6 expression also increases in aging brain and blood ves- sels, consistent with blood IL-6 (see above). Most genes increased during aging are also rapidly induced in young mice by LPS (Terao et al, 2002). These indications of similar inflammatory changes in multiple tissues during aging imply a coordinated pattern, which may involve a small number of tran- scription factors. The NF-κB transcription factors form multimeric complexes with one or more of the five Rel family proteins. These proteins are activated by cell oxidative stress (redox) and regulate hundreds of genes important to Alzheimer disease, cancer, and vascular disease by influencing inflammation and cell death (apoptosis) (Li, 2005; Mattson and Camanadola, 2001; Monaco and Paleog, 2004; Vater et al, 1992). For example, oxidized LDL has biphasic effects on NF-κB in vascular endothelia, which, in turn, influence expression of genes encoding adhesion molecules and scavenger receptors (Robbesyn et al, 2004). NF-κB and other redox sensitive transcription factors could be fundamental to inflammatory cascades of aging. The progressive activation of inflammatory genes during normal aging may have a direct role in impairing gene expression for neuronal functions (Blalock et al, 2003; Blalock et al, 2004; Lu et al, 2004b). In human brain during normal aging, DNA damage was found in the promoters of certain genes with decreased activity during aging (Lu et al, 2004b). Because the DNA is extracted from whole brain regions, we do not yet know the cell types involved that might inform on the source of the oxidant damage. Because other DNA damage is attenuated by diet restriction (Section 3.2), it is plausible that inflammatory processes are involved at some level. Inflammatory gene expression in aging is also found in the fly model, with major induction of genes encoding 20 anti-microbial peptides and other factors of innate immunity (Landis et al, 2004) (Fig. 1.24). The microbial load increases in aging flies (Section 5.6). Moreover, oxygen exposure of young flies induces subsets of the aging changes in gene expression. Thus, we see a remarkable generality in the convergence of inflammation, oxidative stress, and aging 108 The Biology of Human Longevity
  • InflammationandOxidationinAgingandChronicDiseases109 TABLE 1.8 Inflammatory Gene Expression in Aging: Fold-increase During Aging in Rodents Brain Coronary Gastrocnemius Gene Neocortex Cerebellum Hippocampus Artery Liver Muscle Myocardium apolipoprotein E 3a 2c complement (C) 2a 4a 1.3b –2c,d 2g 4-13i C1q a, b, or c C3 >10d C4 5a 4a 1.5b –2c 2h cathepsin S 1.5a 4a 2c 1.5a CD68 (macrophage marker) 2a 4a lysozyme P 2a 6a 2g serum amyloid A (SAA) 3h serum amyloid P (SAP) 2g Interleukin-1 (IL-1) 3e Interleukin-6 (IL-6) 1.3j 1.3j 1.3j 3e tumor necrosis factor (TNFα) 3e numbers >1.5 are rounded to the next integer; same GenBank number as the adjacent leftward entry; a. Mouse, C57BL/6NNia, m; 5, 30 m; Affymetrix (Lee et al, 2000); b. Mouse, C57BL/6 NNia, m; 2, 15 m; Affymetrix (Verbitsky et al, 2004);c. Mouse, C57BL/6 NNia, m; 3,12,18,24 m; Affymetrix (Terao et al, 2002); d. Rat, F344, m; 4,14, 24 m; Affymetrix (Blalock et al, 2003); e. Rat, F344,m; 3,26 m; GEArray , PCR (Csiszar, 2004); g. Mouse, C3B10F1, f; 7,27 m; Affymetrix (Cao et al, 2001); h. Mouse, C57BL/6NNia, m; 5, 30 m; Affymetrix (Lee, 1999); i. Mouse, ‘sedentary’, m; 20, 35 m; Affymetrix (Bronikowski et al, 2003); j. Mouse, BALB/c, m; 3-6,24 m; PCR, ELISA (Ye et al, 1999; Godbout et al, 2004).
  • 110 The Biology of Human Longevity (Queries I and II, Section 1.1) that appears independently in at least one invertebrate and in representative mammals. Shared gene regulatory systems may be sought that will identify ancient ”kernels’ of genetic machinery that modulate outcomes of aging in aerobic organisms. Chapter 2 considers the possibility that deterioration of the gut allows leakage of endogenous pathogens as a cause of systemic inflammation. Lastly, these microarray data bring closure to a major long-standing contro- versy about gene function during aging. In past decades, many presumed that gene functions degenerated globally during aging (Cutler, 1975; Strehler, 1977). However, all the evidence points to high selectivity in the direction and degree of changes in gene expression during aging. Microarray profiling shows that rel- atively few RNAs are altered during aging: <5% of the RNAs detected changed up or down by >2-fold (Table 1.8). These results confirm our earlier work, in which RNA-driven hybridization showed that brain polyribosomal mRNA had the same ‘sequence complexity’ (estimate of numbers of active genes) in young and old rats, within a margin of 5% (Colman et al, 1980). Protein patterns also show the specificity of aging changes. Enzyme activity changes are highly selec- tive (no changes in 80% of enzymes in liver and 88% in kidney) (Finch, 1969; Finch, 1972a). Another approach evaluated rates of protein synthesis in tissues of young and old mice that were injected with differently radiolabelled leucine: [3 H]-young, [14 C]-old (Gordon and Finch, 1974). After co-electrophoresis of sol- uble proteins from both ages, the gels were sliced. The ratios of 3 H: 14 C on 1 mm gel slices showed selective age changes in liver and brain regions (hippocam- pus, striatum, hypothalamus). Each tissue had a few peaks of radioactivity down the electrophoretic lane that differed by age, indicating selective shifts in pro- tein synthesis consistent with selective shifts in mRNAs. Although most somatic cells retain their characteristic phenotypes at the micro- scopic level in tissues, exacting analysis of individual cells by Jan Vijg and TABLE 1.9 Functional Categories of Aging Changes in Gene Expression from Microarray Profiling Braina Liverb Gastrocnemiusc Myocardiumd Higher Lower Higher Lower Higher Lower Higher Lower inflammation- 25% 5 40 15 1 immune stress response 17% 25 16% 8 1 energy 0–5 13 apotosis, 15 25 proliferation macromolecular 9 biosynthesis and turnover a. mouse (Lee et al, 2000); b. mouse (Cao et al, 2001); c. mouse (Lee et al, 1999); d. mouse sedentary (Bronikowski et al, 2003).
  • Inflammation and Oxidation in Aging and Chronic Diseases 111 colleagues is pointing to increasing somatic cell diversity through quantitative variations in specific mRNA content and DNA rearrangements. For example, analysis of mRNA in single cardiomyocytes showed that aging increased cell het- erogeneity in expression levels, e.g., the myosin gene (myl2) mRNA, in contrast to relative invariant expression of the mitochondrial cox-1 (Bahar et al, 2006). Aging myocytes differed by showing both increases and decreases in myl2 mRNA. The role of selective DNA damage may be suspected here, as shown for promoter DNA damage in association with decreased certain mRNAs in the aging brain dis- cussed above (Lu et al, 2004). Moreover, chromosomal DNA rearrangements increase with aging in myocardium and liver (Suh and Vijg, 2006). While these 1 3 5 7 9 11 13 15 17 Young O2 Old PM-bg PMonly PM-MM −1 0 1 2 3 4 5 FBgn0010385: Defensin Chip effect B Oxygen Old 108 234 Protease (17) Transporter (11) Cyt p450 (7) 168 Immune (12) 494 Ox Phos (42/63) ATP synth (9/15) TCA cycle (13/32) Transporter (14) 97 Immune (11) HSP's (9) Antioxidant (6) Cyt p450 (4) Purine P'way (10) 154 Protease (41) Alk Phos (8) Triaclyglycerol Lipase (4) FIGURE 1.24 Aging and anti-microbial gene induction in the fly Drosophila melanogaster. A. 5-fold to 100-fold induction of anti-microbial genes, e.g., attacin (anti-Gram-negative bacteria) and dro- somycin (anti-fungal). (From Landis et al, 2004.) B. Overlap of genes increased during aging and gene induced in young flies exposed to oxidative stress. (Redrawn from Landis op. cit.)
  • 112 The Biology of Human Longevity changes are described as stochastic at the cell level, they may still be driven by systemic physiological shifts (e.g., muscle mitochondrial DNA deletions during aging are increased by hyperglycemia (Liang, 1997) and decreased by diet restric- tion) (Aspnes, 1997) (Section 3.3.2). Thus aging tissues become an increasing mosaic of diverse genomic alterations with a still-to-be-defined impact on cell physiology. 1.9. SUMMARY In most chronic diseases of aging, oxidative stress and inflammation are promi- nent. Moreover, many tissues without specific pathology show modest inflam- matory changes during aging share major subsets of those in chronic diseases of aging. The strong trends for increased blood inflammatory markers (CRP, IL-6) in many studies of aging may be mainly driven by vascular disease. Evidence for the role of insulin/IGF-1 pathways in many pathological processes (vascular dis- ease, obesity, Alzheimer disease) seems broadly consistent with the mutations that increase longevity in experimental models (Fig. 1.3A). Major questions are the role of the age-accumulated bystander damage to proteins and lipids in the inflammatory processes (Query I) and the role of inflammation in further bystander damage (Query II). The case may be strongest for vascular disease that is attenuated by drugs with anti-inflammatory activities considered in Chapter 2. Environmental influences through infections and inflammogens (Chapter 2) and diet (Chapter 3) will evaluate Query III: that diet and environmental pathogens influence diseases with inflammatory components through bystander damage. Alzheimer disease may prove to be as multi-factorial as vascular disease.
  • 113 CHAPTER 2 Infections, Inflammogens, and Drugs 2.1. Introduction 114 2.2. Vascular Disease 114 2.2.1. Historical Associations of Infections and Vascular Mortality 114 2.2.2. Modern Serologic Associations 115 2.3. Infections from the Central Tube: Metchnikoff Revisited 121 2.3.1. Humans: Leakage from Periodontal Disease and Possibly the Lower Intestine 121 2.3.2. Worms and Flies as Models for Human Intestinal Microbial Intrusion 125 2.4. Aerosols and Dietary Inflammogens 126 2.4.1. Aerosols 127 2.4.2. Food 129 2.5. Infections, Inflammation, and Life Span 131 2.5.1. Historical Human Populations 131 2.5.2. Longer Rodent Life Spans with Improved Husbandry 136 2.6. Are Infections a Cause of Obesity? 142 2.7. Inflammation, Dementia, and Cognitive Decline 143 2.7.1. Alzheimer Disease 143 2.7.2. HIV, Dementia, and Amyloid 145 2.7.3. Peripheral Amyloids 147 2.7.4. Inflammation and Cognitive Decline During ‘Usual’ Aging 147 2.8. Immunosenescence and Stem Cells 150 2.8.1. Immunosenescence and Cumulative Exposure 150 2.8.2. Immunosenescence and Telomere Loss 152 2.8.3. Inflammation and Stem Cells 153 2.9. Cancer, Infection, and Inflammation 154 2.9.1. Helicobacter Pylori and Hepatitis B Virus 154 2.9.2. Smoking and Lung Cancer 156
  • 2.10. Pharmacopleiotropies in Vascular Disease, Dementia, and Cancer 158 2.10.1. Anti-inflammatory and Anti-coagulant Drugs 158 2.10.2. Aspirin and Other NSAIDs 161 2.10.3. Statins 162 2.10.3.1. Vascular Disease 162 2.10.3.2. Dementia 164 2.10.4. Sex Steroid Replacement (Hormone Therapy) 165 2.10.5. Plant-derived Micronutrients and Neutriceuticals 169 2.11. Summary 172 114 The Biology of Human Longevity 2.1. INTRODUCTION Chapter 1 emphasized the role of inflammatory processes in vascular disease, from early beginnings before birth. Alzheimer disease shares many of the same inflammatory changes, although cause and effect are less clear. This chapter fol- lows the pathways of Fig. 1.2 further by examining the role of infections and inflammatory agents in vascular disease and Alzheimer disease and selected can- cers. Pharmacologic interventions through NSAIDs and anti-coagulant drugs fur- ther establish the inflammatory mechanisms in vascular disease and may extend to Alzheimer disease. These examples are discussed in relation to Query II (Section 1.1) that inflammation causes bystander damage and Query III that envi- ronmental pathogens and inflammogens influence chronic diseases with inflam- matory processes through bystander damage (Section 1.4). The environmental role in these diverse, slowly developing diseases remains counter-current to traditional thinking, because in general, Alzheimer disease, cancer, and vascular disease are not ‘infectious,’ by Koch’s postulates. That is, with few exceptions for these diseases, infectious agents cannot be isolated, and the disease cannot be transferred and propagated to a test animal. 2.2. VASCULAR DISEASE 2.2.1. Historical Associations of Infections and Vascular Mortality The traditional risk factors for vascular disease (hypertension, obesity, elevated LDL cholesterol, smoking) do not explain about 35% of cases (Section 1.5.3.2). From epidemiologic and pathologic studies, chronic infections may be primary causes or co-factors of inflammation in vascular disease. This controversial concept has been discussed for a century or more (Frothingham, 1911). The hypothesis of inflammation
  • as a co-factor is strongest in human arterial disease, because prodromal microscopic foci of oxidized lipids and activated macrophages are present before birth (Section 1.5.3.1). The evidence for the role of infections in arterial disease, while consider- able and supported by animal models, is still largely circumstantial for humans. Rheumatic heart disease is a classic example of infection-caused heart disease, but with a different etiology than most cardiovascular cases. Until about 1950, rheumatic fever from ‘strep’ infections was still an important cause of damage to heart valves. In particular, streptococcal A substrains cause high incidence of endocarditis and mitral valve scarring (Bispo, 2000; Stollerman, 1997; Wilson, 1940). Rheumatic fever with mitral damage is life-shortening (Jones, 1956). In the 1930s, for example, few survivors of childhood infections lived to age 40, and most died within 15 years of infection (Wilson, 1940, p. 272). Rheumatic heart disease has become rarer in developed countries from public health improve- ments and, then after 1950, the availability of antibiotics. However, heart valves without rheumatic disease often harbor a diverse bacterial flora (see below). Eileen Crimmins and I hypothesize that historical and modern levels of early infection are major determinants of adult vascular disease (Crimmins and Finch, 2006a,b; Finch and Crimmins, 2004, 2005). More generally, historical and modern populations also show associations of infections with later mortality. In some rural parishes of 17th century Sweden, high early infectious mortality was followed by high late life mortality among survivors (Bengtsson and Lindstrom, 2000; Bengtsson and Lindstrom, 2003). Among U.S. Civil War veterans, infectious disease in early adulthood has been associated with heart and respiratory problems after age 50 (Costa, 2000). Cardiovascular disease was twice as prevalent among older Army veterans born before 1845 compared to veterans born in the early 20th century (Fogel and Costa 1997; Fogel, 2004). In Norway 1896–1925, infant mortality, which is a proxy for exposure to infections, correlated strongly with arteriosclerotic deaths 40–69 years later (Forsdahl, 1977) (Fig. 2.1). In the United States 1961–1971, adult cardiovascular disease is also associated with birth cohort levels of infant diarrhea and enteritis (Buck and Simpson, 1982). Other examples are discussed in Crimmins and Finch (2006a). Considered over most of the 20th century, the associations of prior infections on later mortality may explain up to nearly 25% of the decline of both morbid and mortal condi- tions at later ages (Costa, 2000). Relationships of early and later age mortality in birth cohorts are developed further below. 2.2.2. Modern Serologic Associations Stepping forward, we have access to individual histories of infections through persistent antibodies. Serologic associations with cardiovascular disease were first noted in 1987 for cytomegalovirus (CMV) (Adam et al, 1987), soon followed by Chlamydia1 pneumoniae in 1988 (Saikku et al, 1988). Other associations Infections, Inflammogens, and Drugs 115 1 Chlamidophila is the official genus name.
  • 116 The Biology of Human Longevity 600 200 250 300 350 400 450 500 550 40 60 80 100 120 140 200 50 75 100 125 150 175 40 60 80 100 120 140 Men r=+ 0.86 P<0.001 Women Infant mortality 1869−1925, per 1000/Y CADdeathsper100,000/Y r= +0.74 P<0.001 FIGURE 2.1 Past infections and cardiovascular disease in historical Norway. Infant mortality, a proxy for exposure to infections (Finch and Crimmins, 2004, 2005; Crimmins and Finch, 2006a, b), correlated strongly with arteriosclerotic deaths 40–69 years later in Norway 1869–1925. These cor- relations were slightly stronger for men than women. (Redrawn from Forsdahl, 1977.) include the ubiquitous Helicobacter pylori and Mycoplasma pneumoniae; and cytomegalovirus (CMV), hepatitis virus A and -C viruses (HAV, HCV), and herpes simplex virus (HSV-1 and -2) (Belland et al, 2004; Campbell and Kou, 2004; Stassen et al, 2006; Vassalle et al, 2004). Cerebrovascular disease is also associated with C. pneumoniae and H. pylori (CagA strains) (Lindsberg and Grau, 2003). Carotid thickness correlates with antibodies to E. coli endotoxin (LPS) (Xu, 2000), while anti-LPS antibodies correlate with antibodies to oxidized LDL (Mayr et al, 2006). The list grows.
  • In the AtheroGene Study (Mainz and Paris), cardiovascular mortality and coro- nary stenosis were 2–3-fold higher in patients seropositive for ≥ 4 pathogens, rel- ative to those with 0 to 3 seropositivities, with the highest odds ratios for C. pneumoniae and M. pneumoniae (Espinola-Klein et al, 2002a, b; Georges, 2003) (Fig. 2.2A, B). Carotid and femoral artery thickening (IMT) are greater in indi- viduals with chronic infections who also carry proinflammatory alleles of IL-6, IL-1 receptors, and the endotoxin receptor CD-14 (Bruneck Study, northern Italy) (Markus et al, 2006). Blood levels of C-reactive protein (CRP), an inflammatory protein and strong risk indicator of coronary artery disease (Section 1.5, Fig. 1.16B), may also correlate with the number of different seropositivities (Georges et al, 2003; Zhu et al, 2000) (Fig. 2.2C). However, others did not find these serological associations with CRP elevations (Epstein et al, 2000; Lindsberg and Grau, 2003). This is not surprising, because seropositivity often persists long after an infection has subsided and transient elevations of CRP have subsided. Over the life span, the majority of adults become seropositive for C. pneumoniae and CMV (Almanzar et al, 2005; Miyashita et al, 2002). Epidemiological associations of infections and vascular disease are increasingly supported by clinical studies and animal models (Campbell and Kuo, 2003; Coughlin and Camerer, 2003; Libby, 2003; Liuba et al, 2003). C. pneumoniae illustrates sev- eral key issues. This gram-negative bacterial pathogen grows only as an intracellu- lar parasite. Infections typically begin in lungs and may propagate systemically to the vasculature by circulating macrophages. Infections are ubiquitous and reinfec- tions very common (Belland et al, 2004; Campbell and Kuo, 2004; Grayston, 2000). C. pneumoniae is notorious for its broad cell targets, including endothelia, macrophages, and smooth muscle cells of atheromas. It resists antibiotics, which can suppress normal replication without eradicating its effects. Dead C. pneumo- niae still activate the transcription factor NF-κB in endothelial cells, which could promote atherogenesis without active infection (Baer et al, 2003). C. pneumoniae are detected in the majority of atheromas by immunological, genomic, or ultrastruc- tural criteria, but not in healthy arteries (Muhlestein et al, 1996; Shor et al, 1998; Shor, 2001). Heart valves tend to have more C. pneumoniae and other pathogens, in diseased than normal hearts (Juvonen et al, 1998; Nilsson et al, 2005; Nystrom-Rosander et al, 2003). Live C. pneumoniae was cultured from vascular tissues from some cardiac patients (Belland et al, 2004; Campbell and Kuo, 2004). T cells cultured from atherosclerotic carotids were immunopositive in 40% of 17 patients (Mosorin, 2000). These individual variations may arise from successful elim- ination of the pathogen by the host. The suppression of pathogen growth by antibi- otics or cell stress may also add to these variations (Belland et al, 2004; Campbell and Kuo, 2004). However, assay criteria for C. pneumoniae are not well standard- ized and detectability varies from 0–100% (Kalayoglu et al, 2002; Peeling et al, 2000). The high genetic diversity of C. pneumoniae (Belland et al, 2004) may also con- tribute to variability. Some argue that C. pneumoniae and H. pylori in vascular lesions are an epiphenomenon because damaged or necrotic tissues, such as found in vascular plaques, are vulnerable to superinfections (Black, 2003). It is hard to Infections, Inflammogens, and Drugs 117
  • 118 The Biology of Human Longevity Number of seropositivities (Pathogen burden) 0 1 2 3 P<0.0001 Controls CAD cases Percentofsubjects 35 30 25 20 15 10 5 0 B 4 5 6 7 0 A 5 10 15 20 25 No CAD Limited CAD Advanced CAD Mortality(%) Number of seropositivities 6 - 8 4 - 5 0 - 3 FIGURE 2.2 For legend see page 119. prove the causal role of infections in atherogenesis because of their earthly ubiquity— everyone experiences infections (Belland et al, 2004; Campbell and Kou, 2004). Peripheral arteries also show effects of infections. Children with acute respi- ratory infections had impaired regulation of the brachial artery endothelium, by the flow-mediated vasodilation test (Avon Longitudinal Study of Parents and Children, or ALSPAC Study, Fig. 2.3). The effects of infection may have persisted for a year in some individuals (the statistical significance was P<0.06) (Charakida et al, 2005). Longitudinal follow-up continues. Studies of children are valuable because seropositivities are less frequent than in adults.
  • Infections, Inflammogens, and Drugs 119 FIGURE 2.2 Past infections and cardiovascular disease. A. Cardiovascular mortality was 2–3-fold higher in patients seropositive for ≥4 pathogens, relative to those with 0 to 3 seropositivities. Pathogens with seropositive record of past or latent infection detected included C. pneumoniae, CMV, EBV, H. influenzae, H. pylori, and HSV-1, -2. University Clinic Mainz; 1168 Ss. (Redrawn from Espinola-Klein et al, 2002.) B. CAD cases have a higher number of seropositivities: C. pneumoniae, CMV, H. pylori, HSV-1. (Redrawn from Georges et al, 2003.) C. Plasma C-reactive protein (CRP) varies in proportion to the number of different seropositivities. (Redrawn from Zhu et al, 2000). 1.25 1.00 0.75 0.50 MeanCRP,mg/dL Seropositivity 0.25 0.00 3-4Ab (n=116) 5Ab (n=55) <2Ab (n=50) _ p=0.0004 p=0.02p=0.08 C Antibiotics give another test of the infection hypothesis. The large WIZARD trial [weekly intervention with zithromax (azithromycin) for atherosclerosis and its related disorders] is so far inconclusive (Dunne, 2000). Experimental design is diffi- cult, because the treatments may be most effective early during infections (de Kruif et al, 2005). Other long-term studies include the Azithromycin and Coronary Events (ACES) (Belland et al, 2004) and the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE-IT) (Campbell and Kuo, 2004). The first placebo- controlled, double-blind, randomized clinical trial of antibiotics on C. pneumoniae in vascular tissue was inconclusive (Berg et al, 2005). Although 81% of cardiac bypass patients were seropositive, C. pneumoniae DNA was not present in plaques of patients with advanced CAD; antibiotic treatment did not alter seropositivity.
  • Animal models show that infections may have greater synergy with arterial dis- ease when lipids are elevated, as is common during infections (Section 1.4). In rodents, rabbits, and pigs, C. pneumoniae accelerated atherogenesis, but only when the models were made hyperlipidemic (Belland et al, 2004, de Kruif et al, 2005; Liuba et al, 2003a,b,c). Chronic endotoxin also required hyperlipidemia to accelerate atherogenesis (Engelmann et al, 2006). The apoE-knockout (−/−) mouse is an important model, with greatly elevated cholesterol on non-atherogenic diets that promote progressive arterial lesions not found in normal mice. ApoE knock- outs develop aortic plaques by 4 months, followed by vascular rigidity and aneurysms (Wang, 2005, Wouters et al, 2005). Moreover, the lipidemia-induced lesions depend on pathogen-signaling pathways via Toll-like receptors (TLRs) linked to MyD88, an adaptor that activates kinases (Laberge et al, 2005) (Chapter 5, Fig. 5.4). The double apoE and MyD88 knockout mouse had much small aortic lesions, with fewer macrophages and lower chemokines In apoE knockouts, 120 The Biology of Human Longevity FIGURE 2.3 Childhood infections alter arterial endothelial responsiveness. Avon Longitudinal Study of Parents and Children (ALSPAC; population-based study, Bristol UK region) evaluates environmental and genetic influences on health and develop- ment. Vascular endothelial function was evaluated sonographically by flow-mediated constriction (FMD) of the brachial artery diameter. Six hundred Ss aged 10 years were assessed for health; exclusions asthma and chronic infections, or use of antibiotics or anti-inflammatory drugs; acute infections (AI) were 93% upper respira- tory. About 1 year later, there was a reassessment of 50 controls without infection; of 40, prior with acute infection, but without infection since first visit. The AI group FMD response was slightly lower on the second visit (P < 0.06), suggesting a sub- group with persistent endothelial damage. (Redrawn from Charakida et al, 2005.) 20 15 10 5 0 −5 %Arterydiameter Acute infections Controls Visit 1 Visit 2 p<0.001 Visit 1 Visit 2 p=0.85
  • C. pneumoniae caused rapid vascular endothelial damage (aortic contractility, 2–6 weeks after infection) (Liuba et al, 2000). Subsequently, the arterial wall thickens with increased ROS production. The convergence of hyperlipidemia in infection and arterial disease through pathogen-activated pathways suggests that atheroge- nesis is bystander outcome of the indispensable host defense mechanisms. Future case control studies with longitudinal follow-up may be more conclusive. We may learn how to quantify effects of infections on vascular damage by the intensity and duration. There could be a threshold for acute infections of sufficient brevity that do not cause enduring damage. We may anticipate some dose-duration relationships in chronic subclinical infections and arterial disease that are like the ‘pack-years of smoking’ in relation to carotid thickening, as discussed below (Fig. 2.6) and lung cancer. The pathogen burden is indicated by the scaling of vascular event risk to the number of seropositivities, discussed above (Espinola-Klein, 2002; Georges et al, 2003; Zhu et al, 2000). Both research groups use similar terminology, ‘pathologic burden’ (Zhu et al, 2000) and ‘infectious burden’ (Espinola-Klein, 2002), to represent seropositivities, which does not inform on whether infections are active. I suggest the alternative term inflammatory burden to more comprehensively represent these long-term inflammatory influences. Besides infections, the inflammatory burden includes non-infectious inflammogens such as smoke and other aerosols and dietary AGEs produced during cooking (see below). These complexities are well expressed by Stephen Epstein and colleagues: Given that atherosclerosis is a multifactorial disease, Koch’s postulates to establish causality will never be satisfied. These postulates . . . assume a single pathogen, require that all patients with the disease must have evidence of being infected with the casual agent and that all the infected develop the disease. In contrast . . . infectious agents are . . . neither necessary nor sufficient for [vascular] disease development . . . proof of causality can be achieved only in terms of probability rather than as certainty. (Epstein et al, 1999, p. e26). 2.3. INFECTIONS FROM THE CENTRAL TUBE: METCHNIKOFF REVISITED A century ago, Metchnikoff suggested that autointoxication by microbial toxins in the intestinal flora causes chronic poisoning of body cells and premature death (Metchnikoff, 1901, Podolsky, 1998). Recent evidence implicates bacterial leak- age from periodontal disease in vascular disease. Moreover, I suggest the lower gut should also be considered in bacterial leakage, which could be a factor in elevated circulating acute phase proteins during aging. 2.3.1. Humans: Leakage from Periodontal Disease and Possibly the Lower Intestine The mouth normally harbors several hundred bacterial species, mostly as very high density biofilms on teeth. About 10 species of gram-negative anaerobes may be the main pathogens in vascular disease, particularly Porphyromonas gingivalis Infections, Inflammogens, and Drugs 121
  • and Actinobacillus actinomycetemcomitans (Asikainen and Alaluusua, 1993). Their subgingival location is less accessible to antibiotics. We are unavoidably exposed to this high-density flora: Even tooth brushing and flossing can cause transient bacteremia (Carroll and Sebor, 1980, Slots, 2003). The evidence for oral-vascular disease relationships is controversial. In the Atherosclerosis Risk in Communities Study (ARIC Study), severe periodontal dis- ease was associated with thick carotid walls (Fig. 2.4); the effect was greater in men than women (Odds ratio, OR 1.46, range 1.18–1.81) (Beck et al, 2001, 2005; Beck and Offenbacher, 2001). In ARIC (Slade et al, 2003) and other studies, serum CRP, fibrinogen, and IL-6 tended to be elevated in individuals with periodontitis who were otherwise healthy (Chun et al, 2005; D’Aiuto et al, 2004; Schwahn et al, 2004). In atheromas from vascular surgery, nearly half contained DNA from at least one periodontal pathogen (Haraszthy et al, 2000). The periodontitis-vascular association is experimentally supported. In rabbits, periodontitis induced by P. gingivalis increased aortic lipid deposits in propor- tion to the severity of periodontitis (Jain et al, 2003). P. gingivalis generally forms biofilms beneath the gingiva and can invade oral epithelia and vascular endothe- lial cells. Activation of Toll receptors by P. gingivalis is associated with increased IL-1, TNFα, prostaglandin E2 , and leukocyte adhesion molecules (ICAM-1, VCAM-1) (Choi et al, 2005a; Chun et al, 2005; Hajishengallis et al, 2004). 122 The Biology of Human Longevity FIGURE 2.4 Severe periodontal disease is associated with increased carotid artery wall thickness. Atherosclerosis Risk in Communities Study (ARIC Study, Visit 4, 6017 subjects). Means of wall thick- ness: no disease, 0.74 mm; moderate, 0.77 mm; severe, 0.82 mm; most of the statistical difference was in the subgroup with thickness >1mm. (Beck et al, 2001). 35 30 25 20 15 10 5 0 0.5 - 0.6 0.6 - 0.7 0.7 - 0.8 0.8 - 0.9 0.9 - 1.0 1.0 - 1.1 1.1 - 1.2 1.2 - 1.3 None Moderate Severe Percent Carotid artery wall thickness, mm Periodontitis 90th percentile
  • The associations with vascular disease are considered circumstantial (see the analysis of 14 studies) (Kolltveit and Eriksen, 2001), because few studies measured infections by serology or bioassay (Danesh et al, 1999); smoking adds other con- founds (Hujoel et al, 2000). In ARIC, 68% were seropositive for one or more of 17 bacterial species associated with periodontal disease (Beck et al, 2005). High anti- body titers to ≥1 oral pathogen were associated with higher prevalence of cardio- vascular disease, particularly in never-smokers. However, there were no associations of periodontal disease with prevalent cardiovascular disease after adjusting for covariates. Prospective studies should include the individual histories of oral health, smoking, and other lifestyle covariates; multiple time samples of serology for the spectrum of major pathogens; and screening for genetic polymorphisms in IL-1α and IL-1β, which are associated with risk of periodontal disease (Lopez et al, 2005). Diverse microbial ‘communities’ reside in the mouth and lower intestine. The intermediate gut is normally quite sterile, stomach through jejunum (Lin, 2004). The gut epithelial cells in the crypts of Lieberkuhn have tight junctions (zona occludens) that maintain characteristic epithelial cell polarity as part of the barrier to the body cavity and prevent leakage of gut contents (Mullin et al, 2005). Cholera and other pathogenic bacteria alter this vital tight junction barrier. In aging rats, tight junctions become leakier, as assayed by transcolonic epithelial permeability; these aging effects were greater on high fat diets (Mullin et al, 2002) (Fig. 2.5A). The aging gut may have increased leakage, allowing entry of endotoxins into the circulation. As a precedent, CRP elevations are common in inflammatory bowel disease (Poullis et al, 2002). At some threshold, leakage of endotoxins could cause elevations of blood CRP and other acute phase proteins (Section 1.7). The increase of colonic permeability with aging may be due to aberrant crypts (Mullin et al, 2002). Aberrant crypts and villi with cellular dysplasia and loss of epithelial cell polarity increase during aging in the gut of rodents (Mullin et al, 2002) (Fig. 2.5B) and humans (Finch and Kirkwood, 2000, pp. 132–137; Shpitz et al, 1998; Takayama et al, 1998). Stem cell depletion may contribute to these aging changes. Epithelial cells in the crypts of Lieberkuhn proliferate throughout life and are extruded at the tips of the intestinal villi. The crypt stem cells (Potten et al, 2001; Potten et al, 2003) apparently become stochastically depleted during aging (Finch and Kirkwood, 2000, pp. 132–137; Martin, 1998). Radiation and some carcinogens accelerate the loss of stem cells and increase the incidence of abnormal crypts (Magnuson et al, 2000; Martin, 1998). Alternately, TNFα can alter tight-junction permeability via NF-κB activation, as implicated in Crohn’s disease and other chronic intestinal inflammations. Mucosal layer breakdown is not necessary for inflammatory transients to cause gut leakage of potential significance to arterial disease (Lin, 2004). Obesity and diabetes predispose to chronic low-grade infections, which are discussed with effects of diet restriction in Section 3.2.4. The burden of infections (HSV-1 and -2, enteroviruses) shows correlations with insulin resistance particularly in those with C. pneumoniae seropositivity. Thus, metabolic adaptive responses to low-grade infections could be atherogenic by altering insulin sensitivity Infections, Inflammogens, and Drugs 123
  • 124 The Biology of Human Longevity B 0 A 10 20 30 40 50 60 70 6 12 24 M %IncreaseinPBDU-stimulatedflux * ** FIGURE 2.5 Changes in the aging gut. A. Aging increases colonic permeability in male F344 rats. Transepithelial flux of mannitol was induced by phorbol ester (PBDU) in the distal colon in vitro. (Redrawn from Mullin et al, 2002) B. Aberrant intestinal villi in aging mouse: male C57/BL; 5 vs. 30 m. Crypts of Lieberkuhn are 20% decreased; villi are enlarged, with cell loss in the lamina propria. These changes emerge after 12 m and progress further with aging. Directly from (Martin and Kirkwood, 1998). (Continues)
  • Infections, Inflammogens, and Drugs 125 (Fernandez-Real, 2006). Moreover, the sensitivity to low-grade infections may be associated with inflammatory gene variants. 2.3.2. Worms and Flies as Models for Human Intestinal Microbial Intrusion In the worm model of aging, bacterial autotoxicity may be a proximal cause of death during aging (Garigan et al, 2002; Gems and Riddle, 2000; Lithgow, 2003; Mallo et al, 2002) (also discussed in Section 5.2). Wads of bacteria pack the pharynx in aging worms and there is bacterial overgrowth in the pharynx and intestine (Garigan et al, 2002) (Fig. 2.5C)—“. . . the final coup de grace is bac- terial invasion” (Lithgow, 2003, p. 16). The constipation of aging worms may model aspects of human inflammatory bowel syndrome, in which bacterial overgrowth into the normally sterile small intestine causes chronic inflammation (Lin, 2004; Pimentel et al, 2000). Several long-lived worm mutant resist patho- genic bacteria (Garsin et al, 2003) (Section 5.5.2). In the standard culture conditions, worms are fed on live E. coli strain OP50 (Brenner, 1974). However E. coli OP50 may not be the optimum food because life spans may be longer and constipation lessened on diets of some species of yeast (Mylonakis et al, 2002a) or diets of the soil bacterium Bacillus subtilis, which may be more natural foods (Garsin et al, 2003). The concern that live E. coli C Young Old FIGURE 2.5 (continued) C. Gut abnormalities in old worms (C. elegans).The width of the pharynx increases from packed wads of bacteria (black arrowheads), which shows cellular deterioration (white arrowheads). (Directly from Garigan et al, 2002.)
  • 126 The Biology of Human Longevity OP50 is mildly pathogenic is now 40 years old (Croll and Yarwood, 1977; De Cuyper and Vanfleteren, 1982; Hansen et al, 1964): “The longer life span in the absence of bacteria suggests a possible toxicity of bacterial products in the monoxenic cultures” (Hansen and Yarwood, 1964, p. 629). Elimination of live bacteria from the diet increases life spans. A diet of UV- killed bacteria delayed the pharyngeal pack-up of bacteria (Garigan et al, 2002) and increased life spans up to 55%, without loss of fecundity (Gems and Riddle, 2000). Moreover, axenic growth on sterile media supplemented with nutrients doubled the life span (Croll et al, 1977; De Cuyper and Vanfleteren, 1982; Houthoofd et al, 2002; Houthoofd et al, 2004). The axenic cultures maintained the rate of pha- ryngeal pumping to later ages and increased stress resistance, but at the expense of lower fecundity (Croll et al, 1977; Houthoofd et al, 2002). Switching from axenic to bacterial media after larval maturation eliminated the longevity benefit; conversely, raising larvae on bacteria followed by brief antibiotic treatment before transfer to axenic media increased longevity almost as much as growth on sterile media throughout life (De Cuyper and Vanfleteren, 1982). However, these benefits are due not only to the elimination of bacterial toxicity, because this axenic medium was deficient in ubiquinone, a micronutrient obtained from the bacterial diet ( Jonassen et al, 2003; Larsen et al, 2002). These findings raise uncomfortable questions about artifacts from standard lab conditions that are widespread in lab models. I argue that all of our experimental models adapted to the lab should be scrutinized for atypical outcomes of aging. Lab husbandry has eliminated most infections and provides a uniform quality ad lib diet rarely found over the life span in nature. Moreover, our highly inbred lab models were initially selected for early reproduction and high fecundity that may be atypical of the evolutionary background. These concerns will be discussed further in the next chapter in interpreting the obesity common in lab rodents. Flies also show the importance of enteric microbes to aging. In Drosophila melanogaster antibiotics given later in life increased life span by 30% (Brummel et al, 2004). Cell changes in the aging fly gut are consistent with the leakage of gut bacteria later in life. Aging intestinal epithelial cells accumulate virus-like par- ticles (Anton-Erxleben et al, 1983). In the aging housefly (Musca domestica), intestinal cells accumulate concretions (Sohal et al, 1977) and lipid inclusions (Sohal, 1981). Bacteria are seen in sick-looking flies (the insect body cavity is usually sterile) (Flyg et al, 1988). Recent data document the increased bacterial and fungal load of aging flies (Section 5.6.4). The extensive increase of anti- microbial genes during aging in flies (Section 1.8) is consistent with the increased pathogen load of aging flies, possibly from breakdown of the barriers from the gut and exoskeleton. Chapter 5 discusses these and other genetic influences on longevity through inflammation and stress resistance. 2.4. AEROSOLS AND DIETARY INFLAMMOGENS Chronic inflammation is stimulated by intake of non-infectious inflammogens by inhalation and ingestion. These sources have received less attention than
  • infectious pathogens in relation to arterial disease. Airborne inflammogens may be of looming importance to future life expectancy with the accelerating global increases of air particulates (Section 6.4). 2.4.1. Aerosols Aerosols are characterized by size (PM10, <10 µ particle diameter) and composition (mineral, hydrocarbon, sulfur, endotoxin, etc.) and whether the aerosols carry infectious agents (viable vs. non-viable aerosols). Inflammatory responses inde- pendently of infectious agents are induced by airborne inflammogens: Among many examples are smoke from tobacco, fossil fuel, and biomass combustion; dust from agriculture; and endotoxins from feces in the many urban locations with poor sanitation and in livestock and poultry. These sources are pertinent to current aging and to historical improvements in public health (Fig. 1.1A). Smoke is well recognized as a non-infectious (‘non-viable’) aerosol with major consequences to vascular health. Cigarette smoke strongly increases the risk of heart attacks. In the United States in 1990, 20% of deaths from cardiovascular dis- ease are attributable to smoking (Centers for Disease Control and Prevention, 1993). Second-hand smoke is also strongly associated with coronary disease (Zhang and Smith, 2003; Zhu et al, 1997) and lung cancer (Section 2.4.2). Smoking increases the carotid wall thickness in men with dose-dependency (number of pack-years as an estimate of lifetime exposure) (Gariepy et al, 2000) (Fig. 2.6). The mechanisms include proatherogenic increases of oxidized LDL and acute phase proteins. In NHANES III, smokers were twice as likely than non- smokers to have very high CRP (>10 mg/L), with dose responses to the intensity and history of smoking (Bazzano et al, 2003). Second-hand smoke also increased serum CRP into the range of primary smokers and coronary risk in the ATTICA Study (Barnoya and Glantz, 2004; Panagiotakos et al, 2004). Men incur more adverse effects of smoking than women (Fig. 2.6) (Gariepy et al, 2000). Other types of smoke cause chronic lung damage and inflammatory responses consistent with vascular diseases. Common sources of smoke are open combus- tion in fireplaces, furnaces, and factories, which diffuse into the breathing envi- ronment with adverse effects on the lungs (Singh and Davis, 2002). Until the mid-20th century, exposure to wood and coal smoke was almost unavoidable, and still is in many countries (Zhu et al, 1997). ‘Hut lung,’ or domestically acquired particulate lung disease, is associated with inhaled smoke particulates from burning coal, wood, or other fuels and wastes (Gold et al, 2000). Cardiovascular admissions to hospitals were associated with recent exposure to black smoke PM(10) in some studies, e.g., Edinburgh (Prescott et al, 1998). Particulate air pollutants induce vascular endothelial damage (Sandhu et al, 2005; Schulz et al, 2005). Mortality gradients in vascular diseases followed indexes of ambient air pollution in residential zones; not surprisingly, higher income zones had the least exposure to pollution (Finkelstein et al, 2005). Animal models support this epidemiology. Particulate inhalants cause chronic lung damage with lung alveolar macrophage hyperplasia, fibrosis, and accelerated atherosclerosis, Infections, Inflammogens, and Drugs 127
  • e.g., rats exposed to wood smoke (Tesfaigzi et al, 2002) or to fly ash (Schreider et al, 1985). In hypercholesterolemic Watanabe rabbits, exposure to particulate aerosol increased the size of coronary atheromas in proportion to the number of lung macrophages that had phagocytosed particles (Suwa et al, 2002). Dust from corn and grain also induces inflammatory responses (Buchan et al, 2002; Jagielo et al, 1996). Even lab animal bedding materials can con- tain appreciable bacterial endotoxin and (1– >3)-β-D-glucan from bacteria, molds, and plants. When inhaled, these common aerobiosols induce chronic inflammation (Ewaldsson et al, 2002). Humans also experience varying expo- sures to bioaerosols according to occupation and income, which can be a factor in the strong socio-demographic gradients in longevity. Bioaerosols are now a major concern of industrial safety (Burrell, 1994; Menetrez et al, 2001), 128 The Biology of Human Longevity FIGURE 2.6 Smoking increases artery wall thickness in men more than women. Regressions of carotid and femoral intima-media thickness (IMT) on ‘pack-years’ (cumulative smoking dose). (Redrawn from Gariepy et al, 2000.) 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.8 0 10 20 30 40 50 60 70 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.8 0 10 20 30 40 50 60 70 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.8 0 10 20 30 40 50 60 70 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.8 0 10 20 30 40 50 60 70 Men Women CarotidIMT,mmFemoralIMT,mm Lifelong smoking dose (pack-years) r=0.37 p<0.001 r=0.38 p<0.001 r=0.08 p=0.13 r=0.18 p<0.001
  • but are still a hazard of agricultural workers. Workers in sewage plants and garbage collectors also suffer from inhalants that cause chronic systemic inflammation and elevated CRP (Rylander, 1977). Farm workers entering a swine confinement building had rapid elevations of blood complement C3 peaking at 1 h, followed by peak CRP at 2 h (Hoffmann et al, 2003). The smokers in this group had greater responses. Besides airborne live bacteria and fungi, non-viable bioaerosols may contain endotoxins of fecal origin. A specific role of LPS inhalation was shown by the induction of plasma CRP and other inflammatory responses with well-defined dose responses (Michel et al, 1997; Thorn, 2001). The aerosol-vascular disease association is relevant to the historical increase of human longevity during the developments of public sanitation (Section 2.5) and to earlier phases of human evolution as population density increased and encountered increasing exposure to infections, inflammogens, and especially to domestic smoke for cooking and heating (Section 6.2). Genetic risk factors for resistance to domestic smoke and other types of air pollution may have evolved during this time. Curiously, European populations have a high prevalence of a null allele (GSTM1*0) of glutathione-S-transferase M1. GST makes the key anti- oxidant glutathione (Fig. 1.11) and belongs to a superfamily of xenobiotic detox- ifying enzymes with potential importance to human evolution (Section 6.4.2). The M1*0 homozygotes (equivalent to GSTM1 knockout) have impaired lung functions as children and a higher risk of asthma (Peden, 2005). 2.4.2. Food Cooked foods have inflammogens produced by the chemistry of glyco-oxida- tion (Sandu et al, 2005). As discussed in Section 1.4.2, advanced glycation endproducts (AGE) and advanced lipid oxidation endproducts (ALEs) are produced endogenously from chemical reactions of glucose and other reduc- ing sugars with peptide lysine and arginine, which are proinflammatory, atherogenic, and carcinogenic (Kikugawa, 2004; Skog et al, 1998; Vlassara et al, 2002). This saga began in 1912 with Louis Maillard’s discovery of chemical reactions between amino acids and glucose that lead to the loss of lysine and the formation of brownish condensation products (Finot, 2005; Maillard, 1912; Nursten, 2005). These reactions are the basis for browning of foods by broil- ing, or frying, which can increase AGE content 3- to 5-fold (Table 2.1) (Goldberg et al, 2004). AGEs and ALEs are also formed during food processing and storage. AGEs ingested from cooked foods are detected by immunoreac- tivity for the glycation adduct CML (N-carboxymethyl-lysine) (Table 2.1). In healthy adults, plasma CML strongly correlated with the dietary intake of AGE over a 3-fold range (Urribari et al, 2005). The proinflammatory effects of dietary AGEs were directly shown in diet cross-over studies of diabetics (Vlassara et al, 2002). Nutritionally equivalent diets Infections, Inflammogens, and Drugs 129
  • were prepared by different degrees of heating that yielded 5-fold differences in CML. Six weeks on the high AGE diet elevated inflammatory markers, serum C-reactive protein by 35%, and TNFα by 85% in association with 30% higher CML. Similarly, ingested dietary AGEs correlated with serum CML in renal failure patients (Uribarri et al, 2003). Rodents show adverse effects of dietary AGEs. In a mouse model of both atherosclerosis and diabetes (apoE−/− genotype, with STZ-induced diabetes), the high-AGE diet increased aortic lesions, whereas a low-AGE diet decreased lesions below the level in the standard chow diet (Lin et al, 2003). The lesions of the high-AGE diet group had more arterial foam cells and receptors for AGE (RAGE). In another mouse model, 6 months on a high-AGE/high-fat diet induced type-2 diabetes, with impaired glucose regulation and insulin insensitivity (Sandu et al, 2005). The low-AGE/high-fat controls had normal glucose regulation despite similar adiposity. Plasma 8-isoprostane, a marker of lipid oxidation, was increased by the high-AGE diet. As a further scary example, a caramel compo- nent used for coloring beverages (2-acetyl-4-tetrahydroxybutylimidazole, THI) inhibits lymphocyte egress from the thymus by inhibiting the sphingosine 1-phosphate receptor (Schwab et al, 2005). Dietary AGE content may have unrec- ognized influences on rodent studies, because lab chows are typically heated during preparation.2 130 The Biology of Human Longevity 2 Lab chows are typically heated for sterilization and pelleting; however, the details of temperature and duration are not easily known. One ‘low-AGE’ diet had 90% fewer AGEs than the ‘high-AGE’ diet, but this level still could be bioactive (Sandu et al, 2005). Casein, a widely used protein in chows, yields AGEs during industrial prepara- tion (Gilani et al, 2005) (Jing and Kitts, 2002). Speculatively, low-grade, slow AGE side effects may lurk in diets containing casein and other milk-derived products as protein sources. The common nephropathy of aging F344 rats was greatly reduced by feeding on chows containing soy protein versus casein or lactalbumin (Shimokawa et al, 1993b), and median life span was 15% longer (Iwasaki et al, 1988). TABLE 2.1 Advanced Glycation Endproduct (AGE) Content of Common Foods and Effects of Cooking Food CML, kU/g food beef, boiled 1 hr 22 broiled, 15 min 60 tofu, raw 8 broiled 41 milk (pasteurized) 0.05 butter 265 CML (N-carboxymethyl-lysine, an AGE formed by heating); radioimmunoassay (Goldberg et al, 2004).
  • These findings point to an expanding role of dietary AGEs in atherosclerosis and diabetes, and support their designation as glycotoxins’ (Koschinsky et al, 1997; Vlassara, 2005). Vlassara hypothesizes that dietary AGEs, possibly syner- gizing with tobacco and other environmental inflammogens, sustain oxidative stress and chronic inflammation. The AGE receptors (RAGEs) that activate signal- ing pathways with PI3K (Section 1.3.3) may link dietary glycotoxins to longevity pathways that involve insulin/IGF-1 signaling (Fig. 1.3A) and that are also implicated in vascular disease (Fig 1.3B). Besides their color, some Maillard products have definitive tastes and aromas (Schieberle, 2005). Caramel coloring and flavorings have been added to com- mercial foods and beverages for more than a century (Chappel and Howell, 1992; Nursten, 2005). These preferences may have been important in the development of cooking during the last half million years when early humans learned to con- trol fire (Section 6.2). Cooking could have enhanced health by killing parasites and infectious organisms in animal tissues. Moreover, cooking increases the usability of many plants as foods by increasing digestibility and by inactivating toxins that are widely found, e.g., cassava and potatoes. (De Bry, 1994) suggests that early humans used Maillard products as olfactory cues to indicate when tubers with heat-sensitive toxins were sufficiently cooked. Nonetheless, evolution of the omnivorous human diet would have greatly increased exposure to toxins, implying the importance of detoxification mechanisms to enable these new foraging strategies (Sections 3.7 and 6.2.3). Future increases of human longevity may come from better knowledge of these interactions and the mechanisms that remove ingested and endogenously produced AGEs. Dietary changes during human evolution may also have selected for genes that detoxified dietary AGEs, e.g., the recently discovered amadoriases (‘AGE- breakers’) (Monnier and Sell, 2006). Our ancestors ate meat increasingly by a mil- lion or more years ago, a major departure from the plant-based diets of the great apes, and, it is presumed, that of the shared human-chimpanzee ancestor (Section 6.2) (Finch and Stanford, 2004). The more recent use of fire for broiling or roasting meat would have increased AGE ingestion and selected for meat-adaptive genes. 2.5. INFECTIONS, INFLAMMATION, AND LIFE SPAN 2.5.1. Historical Human Populations The recent longevity increases (Fig. 1.1A) also implicate the relationship of infection and inflammation to arterial disease (Finch and Crimmins, 2004, 2005; Crimmins and Finch, 2006a,b). Anonymous reviewers of these papers questioned the importance of vascular disease in deaths before the modern era. However, all evidence points to vascular disease as ancient and ubiquitous “. . . its pattern has always been the same regardless of race, diet, and the Infections, Inflammogens, and Drugs 131
  • stresses of survival” (Magee, 1998, p. 663). The 5,300 year old Tyrolean “iceman” of the Bronze Age evidenced carotid artery calcification (Murphy et al, 2003), which is common in advanced atherosclerosis (Fig. 1.13) (Section 1.5.3.1) and is an independent risk factor of vascular mortality (Doherty et al, 2003; Sangiorgi et al, 1998). Two millennia later, Egyptian mummies of the 18th dynasty preserved calcified arteries and other vascular pathology (Ruffer, 1911). Most large arteries (16/24) in this sample met criteria for atherosclerosis, with half of these specimens showing vascular calcification (9/16). By the European Middle Ages, anatomists were describing arteriosclerosis as “natural to old age” (Long, 1933). Approaching the modern era, the records, scarce as they are, also show cardiovascular disease as a major cause of death in older adult ages. In 19th and mid-20th century England and Sweden, which had low life expectancy, cardiovascular disease was one of the two most important causes of mortality at the oldest ages (Preston et al, 1972; Preston, 1976). For cohorts born after the first decade of the 1800s, the deaths recognized as due to car- diovascular diseases exceed those attributed to infectious conditions. From the earliest date in Sweden, deaths from cardiovascular disease are two times higher than from infectious conditions for those 70–74. For the U.S. Civil War, Fogel and colleagues compared doctors’ reports of heart disease from Union Army veterans aged 65 and over in 1910 versus veterans of the same age in 1983. Heart disease was nearly twice as prevalent in the U.S. Civil War Veterans (76% vs. 40%, age-adjusted) (Fogel, 2004, p. 31). William Osler’s statement from 1892 still holds true today, “Longevity is a vascular question, which has been well expressed in the axiom that ‘a man is as old as his arter- ies.’ To a majority of men, death comes primarily or secondarily through this portal.” (Osler, 1892, p. 664). Moreover, early human ancestors were also likely to incur vascular pathology. Chimpanzees, our closest biological relative, also show extensive hypercholes- terolemia, even on non-atherogenic diets, and die from heart attacks and strokes in captivity (Finch and Stanford, 2004; Steinetz et al, 1996) (Section 6.2). Crimmins and I provisionally conclude that vascular pathology during aging has been prevalent throughout human history and, quite possibly, throughout human pre-history as well (Crimmins and Finch, 2006a). Crimmins and I are evaluating the role of infection and inflammation on later mortality in historical cohorts (Finch and Crimmins, 2004; Crimmins and Finch, 2006a, b). According to our ‘cohort morbidity hypothesis,’ exposure to infections early in life causes chronic infections that, in turn, promote vascular disease, leading to earlier mortality. Tuberculosis, Helicobacter pylori, Chlamydia pneu- moniae, and other gastro-intestinal pathogens noted above are among many chronic infections that have recently diminished. The human environment in rural and urban areas alike was typically filthy by modern standards, with gross continuing exposure to human and animal feces. Running water was not avail- able for convenient washing and bathing. Conditions gradually improved with 132 The Biology of Human Longevity
  • national efforts in public hygiene even before the identification of infectious pathogens and development of immunization at the end of the 19th century. Besides the infectious environment, it was difficult to keep clothes clean and free of ectoparasites, especially before the availability of cheap cotton for clothing, which is easier to wash than wool or leather. Improved nutrition was another major factor in resistance to infectious conditions, due to agricultural improve- ments and the development of national transport systems of canal and rail (Fogel and Costa, 1997; Fogel, 2004; McKeown, 1976). We chose to consider birth cohorts before the 20th century when infections were very common or rampant, but before tobacco smoking became popular. Smoking is a major inflammatory stimulus, as discussed above. Complete birth and death records are available from Sweden from 1751. Sweden also pioneered a national program of inoculation against small pox, begun in 1756, which atten- uated these epidemics by the 1820s, decades ahead of other European countries (Skold, 2000). We also included England (1841–1899), France (1806–1899), and Switzerland (1871–1899). These early cohorts also did not benefit from antibi- otics, which were not widely available before 1950. All the old age mortality examined occurred before 1973. In many countries, dramatic declines in mortality after 1970 are explained best by lifestyle and medical factors. Initial life expectancy was low due to high early mortality characteristic of pre- industrial societies, but increased considerably by 1899 (Fig. 2.7A). The historical trends for declining childhood mortality and old age mortality were remarkably parallel in Sweden, England, and France (Fig. 2.7B). As early age mortality declined, so did later age mortality in the survivors, seven decades later. The increased life expectancy at age 70 was clear in Sweden by 1850 (Fig. 2.7C). These findings support my early estimate that the steady historical increase in the adult age when mortality reaches 1% implies a slowing of aging processes (Finch, 1969, p.12). These associations were tested with regression models for the relationships of temporal change in mortality at ages 70–74 with four childhood stages: infancy, <1 year; early childhood, 1–4; later childhood, 5–9; and adolescence, 10–14 years (Crimmins and Finch, 2006a). Infant mortality is largely attributed to infections. A key feature of this analysis is the comparison of birth cohort, followed throughout life, with the same ages in the corresponding periods. The results are consistent across these four countries: Most of the variance in old age mortality is explained by the early mortality in that birth cohort, 87–96%. The early and later mortality association of cohorts was much stronger than for periods. At a given year, the older adults in a population were born seven decades before the children and had experienced different environments that had a stronger effect on their mortality than in the current environment. Mortality of intermediate adult ages also did not predict old age mortality. The overall mortality curves shift downward quite uniformly as early mortality improves (Fig. 2.7A). Separate analysis of males and females also showed consistent associations in cohorts between early and later mortality Infections, Inflammogens, and Drugs 133
  • (Crimmins and Finch, 2006b). Many specific mechanisms can be considered in cohort morbidity through which recurrent exposure to acute infections or con- tiniued chronic infections accelerates atherosclerosis as well as causing direct damage to heart valves and myocardium (focal lesions and diffuse fibrosis). Extensive immune activation through hyperantigenic stimulation of T cells 134 The Biology of Human Longevity 1 0.1 0.01 0.001 0.0001 0 5-9 15-19 25-29 35-39 45-49 55-59 65-69 75-79 85-89 Y Cohort mortality Mortality/YMortality/Y 1751-60 1931-40 1901-10 1871-80 1811-20 1 0.1 0.01 0.001 0.0001 A 0 5-9 15-19 25-29 35-39 45-49 55-59 65-69 75-79 85-89 Y Period mortality 1751-60 1931-40 1901-10 1871-80 1811-20 FIGURE 2.7 For legend see page 135.
  • Infections, Inflammogens, and Drugs 135 B 16 15 14 13 12 11 10 9 8 7 6 1752 64 76 881800 12 24 36 48 60 72 84 961908 20 32 44 56 68 80 92 Year C LifeExpectancyat70Y FIGURE 2.7 Swedish mortality profiles, based on national records begun in the mid-18th century. From Human Mortality Database (www.mortality.org/Public/main.html). A. Mortality across the life span by cohort and period, plotted on semi-log scale (Finch and Crimmins, 2004). B. Correlations of early and late age mortality by cohorts for Sweden (1751–1899) (a), France (1806–1899) (b), Switzerland (1871–1899) (c), and England (1841–1899) (d) with deviation from maximum cohort height at age 20–21 up to 1899 (measured in millimeters on right axis). (Crimmins and Finch, 2006a). C. Life expectancy at age 70 in Sweden (3-y moving average) showing increases by the mid-19th century, about 70 y after early age mortality had begun to decline (Finch and Crimmins, 2005).
  • 136 The Biology of Human Longevity could also have increased T cell participation in atheroma instability (Section 1.5). Height was also examined because infections slow growth (Section 4.4). The level of early mortality strongly predicted adult height (Fig. 2.7B). In birth cohorts with high early mortality, the survivors were shorter as adults, which we attrib- ute to the greater exposure to infections in childhood. Infections and inflamma- tion cause the reallocation of metabolic resources and energy from growth (Fig. 1.2B), as discussed in detail in Chapter 4. There are also associations of inflammatory genes with fetal growth (TNFα-308 G/G is more prevalent in lower birthweights) (Casano-Sancho et al, 2006) (Section et al 4.10.1). These mechanisms may also account for the progressively decreasing size of adults after 50,000 years ago in human pre-history (Chapter 6, Fig. 6.7). Our model of inflammation in the pathobiology of aging (Fig. 1.2A) also includes important links between maternal infections and inflammation to fetal and infant growth and inflammation. Influenza, malaria, and tuberculosis were common maternal inflammations until recently (Riley, 2001). Malaria and possi- bly other maternal infections can retard fetal growth and increase fetal cytokines (Moormann et al, 1999) (Section 4.5). Smaller babies may have lower resistance to environmental pathogens. These possibilities are not included in the Barker hypothesis of fetal origins that focused on maternal malnutrition as the main cause of the fetal retardation effect on later vascular disease (Barker, 2004), discussed at length in Chapter 4. These manifold effects of inflammation and infection on growth during childhood and on later arterial disease point to a potential unifying theory of human development and aging. 2.5.2. Longer Rodent Life Spans with Improved Husbandry With intriguing parallels to the increasing human longevity discussed above, rodent life spans have nearly doubled in the past 50 y. Elimination of chronic infections through improved husbandry is a major factor. Additionally, arterial and myocardial disease may have been more severe in the early rodent colonies, as indicated for 19th century humans above. Life span increases are best documented for mice of the C57BL/6J (‘B6’) strain, inbred since 1936 at the Jackson Laboratory (Bar Harbor, ME), a pioneering center of mouse genetics (Staats, 1985). B6 males had mean life spans of 18 m in 1948–1956 (Russell, 1966) that gradually increased to the present range of 26–30 m (Finch, 1972; Kunstyr and Leuenberger, 1975) (Fig. 2.9.A, B). Survival curves became increasingly ‘rectangularized’ and right-shifted: Sporadic deaths before 20 m decreased, while maximum longevity increased from 30 to 44 m (Finch, 1969; Tanaka et al, 2000; Turturro et al, 2002). These right-shifts of mor- tality indicate reduction of infections and match those of human populations as health improved (Fig. 1.1A). Unfortunately, the pathology of aging was not well documented for B6 mice during this transition. In modern colonies of B6 mice, the cumulative incidence of cardiomyopathy is about 40% by 24 m (Schriner et al,
  • Infections, Inflammogens, and Drugs 137 10 50 100 0 5 10 15 20 25 30 35 40 M %Survivors Jackson Lab, 1964 Finch, 1971 B FIGURE 2.8 The life span of male C57BL/6 mice has increased progressively since 1957 as animal husbandry has improved. A. Mean life spans from sources cited by (Kunstyr and Leuenberger, 1975): (1) (Russell, 1957); (2) (Muhbock, 1959); (3) (Roderick and Storer, 1961); (4) (Storer, 1966); (5) (Russell, 1966); (6) (Grahn, 1970); (7) (Storer, 1971); (8) (Kunstyr and Leuenberger, 1975); (9) Finch, 1969 (my colony at Rockefeller University and Cornell University Medical School, NYC,1966–1971; sentinel cohort of retired male breeders). B. Survival curves of C57BL/6J male mice, from author’s colony (Finch et al, 1969; Finch, 1972a) and the Jackson Laboratory (Russell, 1966). 17 29 26 23 20 1950 A 19701960 Meanlife-span,M 25 28 27 22 21 19 18 24 1 2 3 4 5 6 7 8 9
  • 138 The Biology of Human Longevity 2005; Turturro et al, 2002) (Section 1.2.2). Rat life spans also increased during the same period: In McCay’s rat colony at Cornell University, where he conducted pioneering studies of nutirion and aging (Chapter 3), mean life span increased from 13 m in 1934 to 20 m in 1943 (McCay et al, 1943). Current lab rat life spans are 26–32 m. Another striking example is the greatly improved longevity of dwarf mice with growth hormone deficiency. Thirty years ago, the Snell dwarf mouse was considered a model for accelerated aging because of short life span (< 6 m) and wizened appearance with gray hair, cataracts, and early onset tumors (Fabris et al, 1972) (Chapter 5, Table 5.3, footnote c). However, in the past decade with improved husbandry, dwarf mice have ‘switched teams’ to become models of slow aging, with life spans over 4 y and delayed onset of tumors. Gray hair is not common in contemporary dwarf mice and, in any case, is not a general trait of aging in B6 mice or other strains (Finch, 1973b). Husbandry improvements that enabled this remarkable transformation probably include reduced infections (see below) and better vivarium temperature control. The general improvements in longevity across all genotypes of rodents in the past decades are not well understood. Even in the early days of laboratory rodent husbandry, some colonies were maintained well enough to achieve contemporary longevity. Slonaker’s rats lived up to age 46 m (Slonaker, 1912) (Chapter 3, footnote 5), while Robertson’s white mice averaged 25 m (Robertson and Ray, 1920) and the Berg-Simms colony females averaged 31 m in the 1960s (Chapter 1; footnote 3, this chapter), discussed below. I suggest that these early colonies were less inbred and closer to wild-types that were recently shown to have greater longevity (Harper et al, 2006a) (Chapter 3, Fig. 3.3). Because the age-related pathology of aging mice at Jackson Labs before 1960 was not reported, we may look to the occasional reports on pathology from other early aging colonies.3 This scattered literature describes conditions in aging rats that may surprise readers. In the 1960s, Wexler and colleagues documented in detail that repetitive mating can accelerate arterial degenera- tion in males as well as females (Wexler and Miller, 1958, 1960; Wexler and True, 1963; Wexler, 1964; Wexler, 1976). These studies employed standard rat stocks (Holtzman, Long-Evans, Sprague-Dawley, Wistar) fed on low fat (4%) diets. Lesions developed in coronary and carotid arteries in proportion to the breeding experience. Heart valve damage was common. ACTH injections 3 Valuable and hard-to-find sources are The Pathology of Laboratory Rats and Mice (Cotchin and Roe, 1967) and The Pathology of Laboratory Animals (Ribelin and McCoy, 1965). Pursuit of this old literature was exasperating. Old books and journals are haphazardly discarded as useless because of their age and stored obscurely or misfiled.
  • or restraint stress also accelerated spontaneous atherosclerosis. Wexler and colleagues postulated that the repetitive mating caused severe stress. It is impos- sible to define the conditions in Wexler’s colony that caused this level of stress during reproduction. Myocardial fibrosis was also associated with the severe coronary artery changes. In current clinical practice, myocardial fibrosis is associated with arrhythmias and sudden death (Siwik and Colucci, 2004; Zannad and Radauceanu, 2005). Moreover, myocardial fibrosis with microscopic scarring was also common in several early rat colonies that may have contributed to premature death. “In older rats fibrosis may be so extensive that it is difficult to understand how the animals remain alive” (Fairweather, 1967, p. 227). Other examples include colonies with 60% prevalence of fibrosis (mild to severe fibrosis) by 20 m (Wilens and Sproul, 1938) and 100% by 20 m (Humphreys, 1957). In modern colonies, myocardial fibrosis is apparently uncommon and arises later (Bronson, 1990). Myocardial fibrosis is associated with inflammation and chronic stress (Holloszy and Smith, 1986), e.g., in rodent models, increased by TGF-␤1 overexpression and decreased by TGF-␤1 deficiency (Brooks et al, 2000; Siwik and Colucci, 2004). Diet-restricted humans have lower myocardial stiffness and plasma TGF-␤1 (Section 3.3.2). Arterial calcification was common in many early colonies but may be rarer today. In the Wilens-Sproul colony, calcification was noted in 46% in pulmonary arteries and 3% in coronary arteries and the aorta (Wilens and Sproul, 1938). In McCay’s colony, aortic calcification occurred in 20% of ad lib fed, but was unex- pectedly 3-fold higher (60%) with diet restriction (McCay et al, 1939). Others described ‘bamboo stick aorta’ with disintegration of the elastic layers and sec- ondary calcification resembling Monckeberg’s medial sclerosis (Fairweather, 1967; Mawdesley-Thomas, 1967; Wilgram, 1959). Sporadic arterial and myocar- dial calcification in aging rats was also reported by (Hummel, 1938; Wilgram, 1959). Moreover, calcification was associated with repetitive breeding in females (Gillman and Hathorn, 1959; Wexler, 1964). Current aging rodents have a low incidence of arterial calcification (<5%) (Bronson, 1990). Arterial calcification is associated with local nodules of Chlamydia pneumoniae in humans (Pierri et al, 2005) and in renal failure (Oh et al, 2002). Coronary artery disease (CAD) was variable in the early colonies. The first report of spontaneous coronary disease on a normal (not fat-loaded) diet may have been from the Wilens-Sproul rat colony, in which 60% had some degree of coronary sclerosis by 24 m (Wilens and Sproul, 1938). Coronary artery stenosis to varying degrees was concurrent with myocardial fibrosis. In the Edinburgh colony, occlusive CAD with intimal plaques was present in 60% of rats by 17 m on a low-fat diet, causing complete blockage of a coronary vessel in some rats (Humphreys, 1957). In another colony, the incidence of coronary stenosis was about 15% (Wissler et al, 1954). CAD was particularly high in female ‘retired breeders’ (Wilgram and Ingle, 1959). However, in two other contemporary colonies, CAD was rare (Berg, 1967; Fairweather, 1967). Infections, Inflammogens, and Drugs 139
  • Three factors may be at work in the increased longevity of laboratory mice, ranked in reverse order of likeliness, in my opinion: genetics, diet, and infec- tious diseases. Improvements at the Jackson Laboratory occurred after 1959 when the Pedigreed Expansion Stock (begun in 1948) was moved to cleaner facilities (Russell, 1966). Longevity increases were not the result of intentional selection for longevity, although routine culling of sickly pups should lower overall mortality by reducing the pool of infections. The lack of correlation between life spans of parents and offspring in these early B6 colonies (Gunther Schlager, in Russell op. cit.) can be considered evidence against genetic drift (crosses of B6 and other strains clearly show inheritance of life span) ( Jackson et al, 2002; Finch and Tanzi, 1997). Cardiomyopathies, nonetheless, may arise more frequently in aging rats maintained on diet restriction with exercise (McCarter et al, 1997) (Section 3.4.2). Dietary fat could be a factor because fatty diets can shorten life span (Chapter 3). The fats fed the first longevity group at Jackson are not known: The composition of the commercial diet was then a ‘trade secret’ (Elizabeth Russell, pers. comm.). In 1959, the Jackson Lab switched to Guilford Chow (11% fat, 19% protein), routinely given breeding females to enhance milk production. Chows with 4–5% fat are currently favored for aging studies (Finch et al, 1969; Turturro et al, 2002). The major change from the 1940s to the 1970s at Jackson and elsewhere was reduction of chronic infections through improved animal and human hygiene. Some reported deaths from epidemic infections as merely ‘accidental’ (Robertson and Ray, 1920). Until the 1970s, laboratory colonies were often infected with microbial infections and skin parasites. Numerous pathogens were gradually minimized or eliminated, including bacteria (Salmonella, Mycoplasma); viruses [coronavirus; ectromelia (mousepox), mouse hepatitis virus, Sendai virus]; and ectoparasites (mites, pinworms) (Bell et al, 1964; Cotchin and Roe, 1967; Flynn et al, 1965; Miller and Nadon, 2000). Early colonies often had chronic respiratory disease (CRD) from endemic Mycoplasma4 recognized by wheezy breathing and crusty noses. While minimizing infections and dietary fat could only increase longevity, we may never know the causes of the extensive myocardial and arte- rial lesions described above. The Wilens-Sproul colony rats had numerous abscesses (‘suppurative lesions’) in brain, ears, genitourinary tract, and lungs (Wilens and Sproul, 1938). 140 The Biology of Human Longevity 4 Chronic respiratory disease (CRD), or catarrh (an ancient name still used), was endemic in rodent colonies up through the 1970s. CRD is characterized by extensive lymphocyte accumulations in the alveolar mucosa, increased mucus secretion and abscesses, and narrowing of the airways and their ultimate collapse (Nelson, 1940) (Nelson, 1963; 1967). I was fortunate to be tutored on CRD by John Nelson (see (Continues)
  • Besides the Jackson Lab mice, another early benchmark rat colony was founded at Columbia University by Benjamin Berg and Henry Simms, which maintained advanced husbandry and exemplary documentation of age-related pathology (Fig. 1.5) (Berg, 1967; Simms and Berg, 1957; Simms and Berg, 1962). Although the Berg-Simms colony was begun in 1945, there was little respiratory disease (< 5% of rats). These rats were not selectively inbred, except to eliminate an ‘eye anomaly’ (Simms and Berg, 1957). Longevity in the Berg-Simms colony was in the range of modern colonies: females, median life span of 31 m and max- imum of 34 m; males, median of 27 m and maximum of 29 m (Berg, 1976). Chronic lesions of aging approximated those of other rat strains in modern colonies (Bronson, 1990) and in the same age ranges: glomerulonephropathy arose before cardiomyopathy and abnormal growths (Simms and Berg, 1957; Simms and Berg, 1962), and arterial calcification was occasional. This health and longevity is remarkable for that time. The current best practice in rodent husbandry is the ‘specific-pathogen free’ (SPF) colony, in which the pathogen load is regularly monitored with sentinel mice (Lindsey, 1998; Miller and Nadon, 2000). SPF status with minimal mycoplas- mas and other pathogens increases fecundity and post-weaning growth and lowers spontaneous mortality (Bell et al, 1964). However, pathogen loads can fluctuate in SPF colonies with agents carried by humans and other adjacent lab animals (Taylor, 1974; Taylor and Doy, 1975). Infections are still embarrassingly common within SPF colonies at major research institutions ( Jacoby and Lindsey, 1997). Although stricter barrier facilities can further reduce transmission of exter- nal infections, the expense and effort are prohibitive. Germ-free (axenic) animals lacking bacteria are problematic for aging studies: Their adaptive immunity is undeveloped, and their flaccid, grossly enlarged caecums develop fatal constric- tions (volvulus) (Gordon et al, 1966). Recent examples also show the importance of animal husbandry. Age-changes in skeletal muscle composition and function observed in ‘dirty’ colonies are neg- ligible in aging rodents from SPF colonies of the same strains (Florini, 1989). Moreover, age-changes in rat liver protein oxidation (carbonyl content) disap- peared in a subsequent cohort of the same strain of rats obtained 10 years later; these differences were confirmed with stored samples (Stadtman and Levine, 2003). Lastly, sporadic hippocampal neuron loss in aging may have Infections, Inflammogens, and Drugs 141 Preface). The most common agent of CRD is Mycoplasma pulmonis, as finally proven in 1971 with germ-free mice (Cassell, 1982; Lamb, 1975; Lindsey et al, 1985; Nelson, 1967; Slauson and Hahn, 1980). Other bacteria can cause CRD (Gay et al, 1972; Lamb, 1975). Mycoplasma are insidiously transmitted in utero and among cage mates; latent infections can erupt in apparently healthy SPF colonies (Lane-Petter et al, 1970), sometimes activated by Sendai virus, another sporadic scourge of aging colonies (Kay et al, 1979; Schoeb et al, 1985).
  • been common in early colonies (Landfield et al, 1977; Meaney et al, 1988) but is not obvious currently (Gallagher et al, 1996; Rasmussen et al, 1996). Variable stress and infections may have been involved. Moreover, early reports of neuron loss (reduced density of large neurons) could be interpreted as neuron atrophy (Fig. 1.7A). In many ways, the hygiene of lab animals parallels that of humans in modern health care: We can minimize childhood infections by immunization and hygiene, but we remain vulnerable to sporadic epidemics. “La pest reste ici” ( J.P. Sartre, The Plague, 1947). 2.6. ARE INFECTIONS A CAUSE OF OBESITY? Viral infections as causes of obesity are being discussed because of evidence that four viruses cause obesity in vertebrate models and serological associations of obesity with infections in humans with obesity and glucose intolerance. These findings should be considered provisional. This story began with the finding that mice developed obesity when infected as weanlings with canine distemper virus (CDV, paramyxovirus closely related to measles) (Lyons et al, 1982; Lyons et al, 2002). CDV causes focal lesions of hypothalamic appetite centers with selective loss of leptin receptors, POMC and cat- echolaminergic neurons in the arcuate nucleus, and hyperplasia of adipocytes and pancreatic islets. Obesity is also induced in other animal models by infections with scrapie (prion disease), Bornavirus, the retrovirus (RAV-7), and AD-36 (human group D adenovirus) (Atkinson et al, 2005; Lyons et al, 2002). In a recent study impressive for its large subject pools, AD-36 seropositivity was 3-fold more prevalent in obese adults (30% than controls, 11%; 502 Ss); moreover, in 28 twin pairs discordant for AD-36, the seropositive individual was fatter than the co-twin (Atkinson et al, 2005). Curiously, AD-36 seropositive individuals had lower cholesterol and triglycerides. In vitro, AD-36 infections of adipocytes decreased increased glucose uptake and leptin secretion; these effects depend on transient expression of viral mRNA, but not viral DNA replication (Rathood et al, 2007). Unlike CDV, no hypothalamic lesions have been found in AD-36 infected mice (Dhurandhar et al, 2000). Another study of obese men who were otherwise healthy found inverse correlations between insulin sensitivity and seropositivity for common infections (HSV-1 and -2, enterovirus, and C. pneumoniae; AD-36 was not included in this panel) (Fernandez-Real, 2006). If the 30% prevalence of AD-36 seropositivity in obesity is generally validated, viral infections may contribute as much to obesity as life style behaviors of eating and exercise, and moreover, may be a causal factor in these behaviors by their impact on the hypothalamus. However, responses to viral infections depend on many factors of host defense, including food intake and exercise (Chapter 3). Resolution of cause and effect in these associations is difficult because obesity and diabetes increase vulnerability to infections (Falagas and Kompoti, 2006) (Chapter 3). Nonetheless, these recent findings support the suggestion of (Lyons et al, 1982) that viral infections have roles in sporadic childhood and adult 142 The Biology of Human Longevity
  • obesity. AD-36 infections that lead to obesity by causing hypothalamic lesions could be another, and milder example, of viruses that propagate by modifying behaviors, although how obesity could particularly favor AD-36 viral propagation is far from obvious. 2.7. INFLAMMATION, DEMENTIA, AND COGNITIVE DECLINE 2.7.1. Alzheimer Disease Infections may also be causes or promoters of Alzheimer disease (AD). This new possibility is even less settled than associations of infections with vascular disease. As a first example, identical Swedish twins who are discordant for dementia may also show long-term outcomes of infections. The first twin to be affected was 3.6- fold more likely to have had periodontal disease (Gatz et al, 2006). Periodontal disease is also associated with infections that interact with arterial lesions (see above), but links of infections to AD are even more speculative. The current (and incomplete) evidence on infections and AD centers on herpes viruses and Chlamydia pneumoniae (Mattson, 2004; Ringheim, 2004; Robinson et al, 2004; Wozniak et al, 2005). Causality is elusive because these infections are ubiquitous. Herpes virus infections occur widely. Postmortem, about 2/3 of all brains, normal and Alzheimer, have HSV-1 (Itzhaki, 1997). HSV-1 infections reside in some brain regions affected by AD (frontal lobe and hippocampus), as well as the trigeminal ganglion. At later ages, HSV-1 infections appear to be active by the presence of HSV-1 antibodies in the cerebrospinal fluid of about half AD and normal controls (Wozniak et al, 2005). Clearly, AD can arise in the absence of active HSV-1 infections! However, in one sample, the apoE4 allele and HSV-1 co-occurred 10-fold more frequently in AD brains than normal elderly (PCR assay). ApoE4 carriers may have a greater risk of neurode- generation from the activation of HSV-1 (Itzhaki et al, 1997). HSV-1 activation in the trigeminal ganglion causes shingles, which afflicts many elderly. A much rarer condition is herpes simplex encephalitis; survivors often have life-long cognitive impairments, possibly from neuronal apoptosis from HSV-1 (Aurelian, 2005). Other candidates are human herpes virus 6 (HHV6) seropositivity in Alzheimer (22% AD vs. 0% controls) (Wozniak et al, 2005) and CMV in vascular dementia (93% VascD vs. 34% controls) (Lin et al, 2001). Some evidence suggests that vaccination against common infections may be protective. In the Canadian Study of Health and Aging, the risk of devel- oping dementia during 5 years was lowered by vaccination for influenza (OR, 0.75), poliomyelitis (OR, 0.6), or diphtheria (OR, 0.41) (4392 Ss, aged ≥ 65) (Verreault et al, 2001). None of these infections is otherwise implicated in dementia. The protective effects of vaccination could be indirect by reduced vulnerability to other infections. Infections, Inflammogens, and Drugs 143
  • The case for C. pneumoniae as a casual factor in AD is less developed than for arterial disease. On one hand, C. pneumoniae persistently infects macrophages and can enter the brain, as in HIV (Stratton and Sriram, 2003). In a culture model, C. pneumoniae promoted monocyte migration across brain endothelia (MacIntyre et al, 2003). Infections of cultured human cerebral microvascular endothelial cells altered the levels of proteins associated with entry of this pathogen into the brain (increased β-catenin, N cadherin; decreased occludin) (MacIntyre et al, 2002). However, the evidence is mixed for the postmortem detection of infections. One study detected C. pneumoniae in cerebral vessels and glia in more Alzheimer brains than controls (Balin et al, 1998; MacIntyre et al, 2003). However, subse- quent studies could not detect C. pneumoniae DNAs even in these same brains (Gieffers et al, 2000; Nochlin et al, 1999; Ring and Lyons, 2000). Vascular demen- tia studies were also mixed: positive association (Yamamoto et al, 2005) versus no association (Chan Carusone et al, 2004; Wozniak et al, 2003). Antibiotic treatment of AD patients with doxycycline and rifampin did not alter seropositivity for C. pneumoniae but, unexpectedly, slowed early cognitive declines (Loeb, 2004). C. pneumoniae isolated from AD brains caused rapid formation of amyloid plaques in a normal mouse (BALB/c) (Little et al, 2004), which, like other ‘wild- type’ mice, never develops plaques during normal aging. Aβ1-42 containing plaques increased during the three months after infection, and a subset of plaques had fibrillar amyloid. Activated astrocytes around the plaques and distant from plaques suggest general inflammatory responses. Neuronal perikarya also had increased Aβ1-42, an early Alzheimer change (Section 1.6.3). The formation of fib- rillar Aβ is puzzling because the mouse Aβ1-42 protein differs from the human in three substitutions that reduce aggregation (Section 1.6.). Nonetheless, mice over- expressing TGF-β1 (an inflammatory factor upregulated in Alzheimer brains) slowly developed fibrillar Aβ deposits around cortical microvessels; these deposits were preceded by thickening of the basement membrane. These changes are absent during aging in wild-type mice (Wyss-Coray et al, 2000). Transgenic AD mice also show acceleration by systemic inflammation from i.p. injections of the endotoxin LPS (Godbout et al, 2005; Konsman et al, 2004; Scott et al, 2004). In the triple transgenic mouse model of AD, which has both amy- loid plaques and neurofibrillary degeneration (Section 1.6), LPS caused earlier hyperphosphorylation of neuronal tau but, contrary to expectations, did not alter amyloid deposits (Kitazawa et al, 2005). In other AD transgenics, LPS induces APP and Aβ in neurons of the cerebral cortex and hippocampus, which are AD brain regions (Sheng et al, 2003). Microglial activation correlated strongly with neuronal APP and Aβ. Aging mice had greater inflammatory responses and more prolonged ‘sickness behaviors’ in response to LPS (Godbout et al, 2005). The effects of aging on responses to LPS were not examined by these transgenic studies, which used rel- atively young mice. In stroke models, LPS pretreatment increases subsequent brain damage from cerebral artery blockade (Becker et al, 2005). Moreover, blood CRP is also elevated by LPS (Section 1.2) and elevated peripheral CRP increased damage from stroke in rats (Gill et al, 2004). Thus, in populations with 144 The Biology of Human Longevity
  • high levels of infection and inflammation, stroke may cause greater brain damage and higher mortality. It is unclear if systemic endotoxins cross the blood-brain barrier. In rodents, systemic LPS binds to Toll-like receptors (TLRs) (Section 1.2 and Section 2.2.2 above) on cerebrovascular endothelia, which may increase vas- cular permeability to some sugars (Singh and Jiana, 2004). Do environmental inflammogens promote AD? Smoking, while a strong risk factor in vascular disease (above), is not a clear risk factor for AD. While some case control studies indicate that smokers had lower risk of AD, recent cohort studies showed higher risk (Letenneur et al, 2004; Luchsinger and Mayeux, 2004; Sabbagh et al, 2005). Occupation and education are confounding variables. Smokers with AD may be younger at death but had the expected neuropathol- ogy (Sabbagh et al, 2005). Lastly, obesity and the metabolic syndrome are associated with chronic, low-grade inflammation (Section 1.7). As discussed in the next chapter, obesity and diabetes increase the risk of infections, while obese mice also have greater neuroinflammatory responses to LPS (Scott et al, 2004). 2.7.2. HIV, Dementia, and Amyloid Dementia with memory loss is common in HIV sufferers with AIDS (acute immunodeficiency syndrome) (Selnes, 2005). The HIV virus enters the brain through macrophages but does not infect neurons with the production of further infectious virions. Diffuse damage and inflammation are found throughout the brain. Cases are often complex because of infections by other pathogens that cause diverse damage, e.g., demyelination (multifocal leukoencephalopathy) from the neurotropic JC virus (Del Valle and Pina-Oviedo, 2006). Recently, diffuse amyloid Aβ deposits were found in the brains of AIDS patients (Green et al, 2005; Rempel and Pulliam, 2005) in proportion to the duration of infection (Fig. 2.10). The average age at death was 43 years, which is two decades before amyloid deposits become generally common. Nearly 50% of 150 brains from AIDS victims had diffuse Aβ deposits in the frontal cortex, most frequently near arteries (Green et al, 2005). Neuronal Aβ was also common, an early change in Alzheimer disease (Section 1.6.3). So far, AIDS brains have not shown two hallmarks of Alzheimer disease: compact neuritic plaques (Rempel and Pulliam, 2005) or neurofibrillary tangles (Green et al, 2005). These findings with well-characterized monoclonal antibodies confirm smaller studies of AIDS brains, which also included younger ages (Esiri et al, 1998; Izycka- Swieszewska et al, 2000; Rempel and Pulliam, 2005). Two mechanisms are note- worthy because of possible synergy: (I) induction of the amyloid precursor protein (APP) in neurons as an acute phase response during the brain inflam- mation of AIDS (Section 1.6.4) and (II) decreased degradation of Aβ by nepro- lysin, an endopeptidase enzymatically inhibited by Tat peptides from the HIV-encoded protein (Daily et al, 2006; Rempel and Pulliam, 2005). Both mech- anisms should increase the production of oligomeric and solid Aβ. More infor- mation may be anticipated from HIV carriers who did not develop AIDS because of successful anti-viral therapy. Infections, Inflammogens, and Drugs 145
  • 9 8 7 6 5 4 3 10 2 2 4 6 8 10 Length of infection (years) 12 14 16 18 Aβplaques/section r=0.562 P<0.05 CONTROL HIV-1 AD A B C D E F G H FIGURE 2.9 For legend see page 147.
  • 2.7.3. Peripheral Amyloids Tissue amyloid deposits (amyloidosis) are associated with chronic infections, e.g., tuberculosis (de Beer et al, 1984, Sipe, 1994, Urban et al, 1993). Peripheral tissue amyloid accumulations are also increased by endemic bacterial flora and common endemic pathogens in mice. Specific-pathogen free mice of several genotypes had no tissue amyloids (thioflavin-binding, not otherwise character- ized) up through 28 m (mean life span), whereas ‘conventional’ (dirtier) colonies had amyloid deposits (Lipman et al, 1993). In mice transgenic for mutant human transthyretin (TTR), the amyloid deposits of the gut and the penetrance of the mutant TTR-induced peripheral neuropathy (polyneuropathy) were strongly influenced by the microbial flora (Noguchi et al, 2002). TTR amyloidosis was increased by exposure to enterobacteria and yeast, and decreased by anaerobic cocci. Social stress or social deprivation may also influence amyloidosis, e.g., more amyloid accumulated during aging when mice were housed in groups versus solitary (Lipman et al, 1993). 2.7.4. Inflammation and Cognitive Decline During ‘Usual’ Aging Elevations of CRP and other acute phase proteins are common at later ages in populations (Fig. 1.23) and may be modest risk factors of cognitive declines. The MacArthur Study of Successful Aging followed high performing elderly for 7 years in a carefully controlled analysis (Weaver et al, 2002). Elderly in the high- est IL-6 tertile had twice the risk of cognitive decline (Fig. 2.10A). This subgroup may be < 25% of all elderly with elevated IL-6. Other studies show similar risks. In the Health, Aging, and Body Composition Study of well-functioning elderly (African American and Caucasian) (Yaffe et al, 2003), during 2 years, the top tertile of inflammatory markers showed a risk of cognitive declines in association with levels of CRP (OR 1.41) and IL-6 (OR, 1.34), but not for TNFα. During 5 years of observation, 23% had significant cognitive decline. Subgroups with both the metabolic syndrome (Chapter 1) and high inflammatory markers had 1.66-fold higher risk of cognitive impairment (Yaffe et al, 2004). In the Helsinki Aging Study over 5 years, the risk of cognitive decline increased with CRP > 5 mg/L (OR, 2.32) and diabetes (OR 2.18) (Tilvis et al, 2004). Infections, Inflammogens, and Drugs 147 FIGURE 2.9 AIDS induces Aβ-containing ‘diffuse plaques’ in frontal cortex sections with increasing frequency during the disease (Rempel and Pulliam, 2005). Average age 43 y (33–58). Immunostained with monoclonal 6E10, panels (a–e). Panel (a) HIV-1 seronegative control, age 46 y; (b, d, f) HIV-1 seropositive, 48 y; (c, e, g) Alzheimer disease, 84 y. Arrows, Aβ plaques in (b) and (c). Panels (d) and (e), Bielschowsky silver staining for neuritic plaques, negative for plaques from HIV-1 (f) and positive for plaques from Alzheimer (g). (h) Correlation of Aβ load versus duration of HIV-1 infection (r, 0.56, P <0.05).
  • 148 The Biology of Human Longevity −49 −40 −30 −20 −10 −7 0 10 21 3 (high) Cognitivechange 1 (low) 2 (mid) IL-6 Tertiles A MacArthur Study of Successful Aging FIGURE 2.10 Elevations of acute phase proteins predict cognitive decline during aging. A. Plasma IL-6 elevations are modest predictors of cognitive decline in healthy elderly during observation over 7 y. (Redrawn from Weaver et al, 2002.) From the MacArthur Study of Successful Aging; 1,189 Ss, selected as a high-functioning sample of NHANES, age 70–79 y; assessed by ‘Portable Mental Status Questionnaire’ of delayed recall and the MacArthur Battery in the home for cognitive and physical evaluation. In the initial sample, IL-6 varied inversely with cognitive scores. In the 7-y follow-up, 0 1 2 3 4 5 6 1st 4th3rd2nd High-sensitivity C-reactive protein (quartile) All dementia Honolulu-Asia Aging Study Alzheimer's Disease AD with CVD RelativeRisk B (Continues)
  • Infections, Inflammogens, and Drugs 149 However, the Longitudinal Aging Study (Amsterdam) found mixed associa- tions: Cognitive decline was associated with elevated α1 -antichymotrypsin (ACT), but not with elevations of CRP or IL-6 (Dik et al, 2005). In the Maastricht Aging Study (MAAS) for those over 50 during 6 years, elevated CRP was asso- ciated with decline in only one cognitive test (word learning) (Teunissen et al, 2003). The InCHIANTI study of aging in Tuscan communities documents the age-related increase of IL-6 and other acute phase proteins (Cesari et al, 2004) (Fig. 1.21) and may soon report on cognition. Overall, the associations of acute phase elevations and cognitive declines are modest and may not generalize between populations. Some divergences may be due to different tests used. But, a larger question lurks. Do the cognitive declines in “normal aging” represent incipient dementia from Alzheimer or cerebrovascular disease? Although these studies of normal aging excluded individuals with signs of dementia at entry, none was designed to identify the early, preclinical stages (CDR 0.5 or mild cognitive impairment) (Section 1.6.3). However, the Honolulu-Asia Aging Study of men clearly shows associations of blood CRP at middle age with Alzheimer and vascular dementia 25 years later (Schmidt et al, 2002). Relative to the lowest quartile, the top three quartiles showed 3-fold higher risk of Alzheimer and vascular dementias 25 years later (Fig. 2.10B). The risks did not scale smoothly with CRP levels, but are highly significant for each of the top three quartiles: for Alzheimer, for vas- cular dementia, and for mixed cases. These associations were independent of cardiovascular disease in this sample, which may represent selective mortality. However, InCHIANTI attributed most of the elevations of CRP and other acute phase proteins to cardiovascular disease (Cesari et al, 2004). Seropositivity to C. pneumoniae was prevalent among the elderly and correlated with IL-6 and TNFα (Blanc et al, 2004). As noted in Section 1.5.4, vascular and Alzheimer disease share many risk factors including elevated acute phase proteins, ele- vated cholesterol, and hypertension. FIGURE 2.10 (continued) cognitive scores decreased in 33.6%. The change in cognitive score (verti- cal axis) over 7 y grouped by tertiles of initial IL-6, left to right in ascending order (tertiles 1,2,3). The Box plots show ± bars’ of the 1.5 interquartile range; o, outliers. The horizontal line is the cut-off of significant decline. B. Plasma C-reactive protein (CRP) in the Honolulu-Asia Aging Study (males only) in 1975 showed associations of blood CRP at middle age with Alzheimer and vascular dementia 25 y later. CRP values were stratified as quartiles. OR is odds ratio of dementia risk, adjusted for possible confounders. (Redrawn from Schmidt et al, 2002.)
  • 150 The Biology of Human Longevity 2.8. IMMUNOSENESCENCE AND STEM CELLS Immune system aging is very complex: Innate immunity tends to increase, exem- plified by arterial macrophages and local inflammation, whereas adaptive immu- nity tends to develop restriction of repertoire from oligoclonality and depletion of T cells (Sections 1.2 and 1.5.1). Inflammatory processes may interact with aging in instructive immunity and stem cell generation. 2.8.1. Immunosenescence and Cumulative Exposure The thymus is highly sensitive to atrophic changes during acute infections, which alter the critical micro-environment and impair thymocyte proliferation (Savino, 2006). During the life span, humans and other mammals show major shifts from naive T cells to memory T cells with progressively restricted repertoire (oligo- clonality) (Sections 1.2.2 and 1.4.3). This aspect of immunosenescence can be accelerated by greater exposure to antigens. The ‘hyperstimulation hypothesis’ of naive T cell depletion is most strongly developed for the very common infections by cytomegalovirus (CMV). Deficits of memory cells and the presence of highly differentiated T cells with CMV-specificity are strongly linked to CMV seroposi- tivity. These changes characterize an ‘immune risk phenotype’ with higher mor- tality in the elderly, observed in two European populations (Akbar and Fletcher, 2005; Huppert et al, 2003; Pawelec et al, 2005; Wikby et al, 2005). Continued antigenic stimulation is hypothesized to drive clonal expansions of memory T cells and is predicted to eventually clonally deplete cells with the highest anti- genic affinity. Moreover, the hyperstimulation by one antigen can have bystander effects that activate other T cell specificities due to local secretion of IFNα and TNFα (Fletcher et al, 2005) (Section 1.4.3). Although CMV is a ubiquitous infection, in 60–100% of adults depending on the population, nonetheless, some individuals reach old age without becoming seropositive. By comparison with CMV-seronegative individuals aged 22–91, healthy CMV-seropositive individuals with latent infections at all ages had 25–50% fewer naive T-cells and an increase of differentiated memory T cells (CD8+ CD28− effector cytotoxic) in all age groups, particularly the older (Almanzar et al, 2005). Moreover, IFNγ expression is higher in CD8+ T cells with CMV-seropositivity, which would increase the body load of this powerful cytokine and possibly syn- ergize with inflammatory processes throughout the body. The hyperstimulation hypothesis of immunosenescence predicts that popula- tions with chronic infectious disease should show premature T cell senescence. HIV is being examined from this perspective. Young HIV carriers have impaired T-cell responses to vacci-nation for preventing childhood infections; e.g., measles, polio, and influenza antigens induce weaker responses (Rubinstein et al, 2000; Setse et al, 2006). HIV-specific T cells have lower proliferation and cyto- toxicity, both as found in immunosenescence of the elderly (van Baarle et al, 2005). Moreover, HIV patients show immune impairments to other antigens.
  • Multiple immunizations with a T cell-dependent neoantigen (φ174, non-infectious bacteriophage) caused progressively smaller responses, unlike healthy controls, presumably because of the already limited pool of naive T cells; booster immu- nizations with φ174 also increased plasma HIV viremia (Rubinstein et al, 2000). These findings support the hyperstimulation hypothesis of immunosenescence. These effects extend to antigenic stimulation by intestinal nematodes (helminth parasites). In Ethiopians infected with HIV and nematodes, the plasma HIV load was correlated with the helminth load; successful de-worm- ing treatment also decreased HIV viremia in 8/11 patients (Wolday et al, 2002). Ethiopian immigrants to Israel have given unique opportunities to study the reversibility of age-like immunological changes after leaving a highly infec- tious environment (van Baarle et al, 2005). About 60,000 Ethiopians of all ages arrived in two waves, 1984–1985 and 1991. Immigrants were often infected with worms, but not with HIV, malaria, or TB. The immigrants were charac- terized by abnormally low proportions of naive CD4+ T cells and excess T memory cells (‘broad T cell activation’) (Borkow et al, 2000; Kalinkovich et al, 1998; Kalinkovich et al, 2001). Nearly half had inverted CD4:CD8 ratios <1, not found in control groups that resemble the ‘immune risk phenotype’ of elderly northern Europeans (Section 1.2). T cell impairments included decreased acti- vation of ERK2 kinases, β-chemokine secretion, and delayed type hypersensi- tivity (DTH, memory T cell dependent). Effective de-worming restored the DTH (Borkow et al, 2000), normalized the ratios of CD4:CD8 T cells, and almost normalized the T cell activation profile (Kalinkovich et al, 1998). Evidently, elimination of the nematode antigen load partly regenerated a naive T cell pool, as occurs in HIV infections. Even when not infected with HIV or worms, by the age of 5, Ethiopians have abnormally high proportions of memory T cells, indicative of antigenic hyperstimulation (Tsegaye et al, 2003). Nonetheless, their neonatal T cell proportions were normal, suggesting effective placental barriers to maternal infections (see Section 4.6.4). Their short life expectancy (about 40 y, like other pre-industrial populations, Fig. 1.1 legend) and high memory T cell profile suggest that some Ethiopians are exposed to condi- tions prevailing in 18th century Europe where life span was also about 40 y (Section 2.4). These continuing studies promise unique insights on the rate of reversibility of immunosenescence after the transition to a 21st century environment with greatly improved health care, hygiene, and nutrition. Another population in transition is the Tsimane of the Bolivian Amazon basin. These forager-horticulturalists have had limited access to modern medicine and manifest a high incidence of parasites and other infections (Gerven et al, 2007; McDade et al, 2005). Their short life expectancy (43 y, about the same as the Ethiopian immigrants above) and age-specific mortality profile resemble mid-19th century Sweden (Fig. 1.1B, Fig. 2.7A). Blood CRP elevations are consistent with their high pathogen load: By age 40, the Tsimane have had as many years with high CRP as those in the United States by age 60. Immune cells have not been characterized. Other populations of interest for accelerated immunosenescence Infections, Inflammogens, and Drugs 151
  • are Gambians, who have seasonal cycles of infections that alter T cell functions (Section 4.6.2) (Moore et al, 2006), and rural Guatemalans, who received nutri- tional supplements and health care in controlled studies (INCAP, Section 4.2) (Scrimshaw, 2003). Mice also accumulate memory T cell clonal expansions in response to chronic antigenic stimulation, e.g., by HSV-1, which induces a strong cytotoxic lympho- cyte response (Messaoudi et al, 2006). Unexpectedly, these memory T cells were not antigen specific and were induced just as effectively by the immune adju- vant, suggesting important by-stander effects that increase T cell clonal expan- sion (Section 1.4.3). Moreover, normal intestinal flora also influence memory T cell clonal expansions, as shown with germ-free mice (Kieper et al, 2005). We may conclude that the individual profile of aging in instructive immunity is closely linked to the cumulative antigenic experience that certainly begins at birth with the first full encounter to the germy world and may well begin earlier in the undocumented levels of prenatal infections. Internal sources of antigens also extend far beyond enteric and oral fauna to auto-antigens, possibly includ- ing AGEs accumulated on long-lived proteins. Lastly, the trends for increased levels of proinflammatory cytokines may also influence adaptive immunity, because IL-1, IL-6, IL-18, etc., modulate many aspects of immune progenitor cell differentiation, including stem cells. 2.8.2. Immunosenescence and Telomere Loss Chronic inflammation without infections can cause telomere shortening. Chronic smokers, for example, have shorter telomeres in peripheral blood lymphocytes (Valdes et al, 2005) and less telomerase (hTERT) (Getliffe et al, 2005). The path- way linking smoking to peripheral lymphocytes may be direct because smoke com- ponents rapidly activate blood monocytes by redox-dependent pathways (Walters et al, 2005). Chronic skin inflammation is also associated with telomere shortening. Children with atopic dermatitis have activated skin T cells; when cultured, skin T cell cultures had 25% shorter telomeres than normal blood lymphocytes (Bang, 2001). In psoriasis, blood T cells also have shorter telomeres (Wu et al, 2000). Chronic psychological stress was correlated with telomere shortening in peripheral lymphocytes in women (Epel et al, 2004). Although these pre- menopausal women were considered ‘healthy,’ this study did not report on recent infections, which would be expected with histories of stress and which could have caused immune cell proliferation independently of perceived psy- chological stress. Stress is well known to increase the incidence of infections in humans (Marsland et al, 2002) and animals (Sheridan et al, 2000). Short telomeres may predict greater mortality and shorter longevity. As noted above, CMV-seropositive individuals had shorter telomeres. Although CMV is considered a benign infection in those with healthy immune systems CMV- seropositivity is associated with higher mortality in the elderly (Pawelec et al, 2005). Again, short telomere length in blood lymphocytes was associated with increased 152 The Biology of Human Longevity
  • mortality at all ages by 2-fold, with 8.5-fold more infections and 3.2-fold more heart disease (Utah blood donors) (Cawthon et al, 2003). Among stroke sur- vivors, those with short telomeres in blood lymphocytes were more likely to develop dementia and die sooner (von Zglinicki and Martin-Ruiz, 2005). The generality of these findings is unclear. 2.8.3. Inflammation and Stem Cells Inflammation may prove to impair stem cell generation and survival. For exam- ple, in the adult rodent brain, neuron stem cell formation (de novo neurogene- sis) was strongly inhibited by LPS endotoxin (direct i.c.v. infusion) (Ekdahl et al, 2003). Neurogenesis was inversely correlated with the density of local microglia. Neurogenesis was restored by minocycline, an antibiotic that passes the blood- brain barrier and suppresses microglial reactions in neurodegenerative disease models. These findings give a rationale for evaluating the role of inflammation in neurogenesis during Alzheimer and Parkinson disease, which have chronic local inflammatory responses, as well as in the milder inflammation of normal brain aging (Chapter 1). Circulating C-reactive protein (CRP) may inhibit generation of vascular endothelial progenitor cells (EPCs), which are implicated in adult vascular repair and regeneration (Section 1.5.3.4). In vitro, clinical levels of exogenous CRP inhibited EPC migration and adhesion; moreover, high CRP also inhibited for- mation of capillary tubules (angiogenesis) and the expression of proangiogenic factors (VEGF, IL-8; eNOS) (Verma et al, 2004; Suh et al, 2004). Other factors are anticipated besides elevated CRP because angina patients showed very modest correlations of CRP and circulating EPCs (r2 = 0.09) (George et al, 2004). Responses to bacterial pneumonia imply further roles of EPC. Elderly patients with bacterial pneumonia, but without clinical cardiovascular disease, had several-fold more circulating EPCs during the acute phase, relative to post- treatment (Yamada et al, 2005). Those with low EPCs before treatment had per- sistent lung fibrosis, which suggests that EPCs contribute to lung repair. Moreover, EPC and total lymphocyte counts were strongly correlated, consistent with the poor prognosis of lymphocytopenic pneumonia patients. This association implies the co-regulation of EPCs with other marrow stem cell populations. In summary, these observations suggest that chronic exposure to infection and inflammogens may drive increased mortality during aging from three causes. (I) The diminished pool of naive memory T-cells truncates the primary immune response to new infections, whereas the highly differentiated state of memory cells limits the strength of the secondary responses. (II) The cytokine shift to increased IFNγ production by differentiated T cells may interact with the proinflammatory trend during aging. (III) Atherogenesis may interact with these processes at many levels, from local reactions of CMV-specific T cells in unsta- ble atheromas to reduced generation of stem cells that may be critical in repair of vascular, lung, and neural tissues. Infections, Inflammogens, and Drugs 153
  • 2.9. CANCER, INFECTION, AND INFLAMMATION Some infections that cause chronic local inflammation also increase the risk of cancer and other abnormal growths. About 15% of all cancers are attributed to infections, directly or indirectly (Herrera et al, 2005). Links between aging, cancer, and endogenous inflammation are also hypothesized (Sarkar and Fisher, 2005). Many cancers and abnormal growths are associated with local inflammatory cell responses that stimulate cell proliferation. In the mutational theory of oncogen- esis, the mutagenic progression to malignancy begins with DNA damage. Mutations accumulate during DNA replication, which may be stimulated endoge- nously, e.g., by sex steroids in reproductive tissues or exogenously, e.g., ultra- violet damage, carcinogens, viruses. Inflammatory processes after initiation are illustrated by the ras pathway. Ras protooncogenes (H-ras, K-ras, N-ras) are often activated by mutations; e.g., 50% of colon cancers have mutations in ras (Bos, 1989). Activated ras protein, in turn, induces IL-1 and IL-8 and other proinflam- matory cytokines (Sparmann and Bar-Sagi, 2004; Liu et al, 2004). IL-8 induction involves ERK-MAPK and PI3K pathways, which also influence longevity (Fig. 1.3). IL-8 mediates the recruitment of macrophages and other inflammatory cells that promote tumor vascularization (angiogenesis). Local inflammatory responses may also produce ROS that induce further mutations on the pathway to malignancy. Several examples are discussed in more detail that represent inflammation from infections (next) and from smoking (following section) and that further illustrate the importance of bystander damage during inflammation (Chapter 1.4). 2.9.1. Helicobacter Pylori and Hepatitis B Virus H. pylori is conclusively linked to inflammatory bowl disease and gastro-intestinal cancers, which rank second among malignancies as cause of death (Herrera et al, 2005; Parkin et al, 2005; Sugiyama and Asaka, 2004). These links are much stronger than associations of H. pylori with vascular disease (Section 2.2). Most humans have submucosal infections of H. pylori: 76% in developing countries, 58% in developed countries (Parkin et al, 2005). Infections are usually acquired early in life, and the risk is several-fold higher if parents are infected (Webb et al, 1994; Rothenbacher et al, 2002). Fortunately, most carriers (85%) are asymptomatic. However, about 15% of carriers develop peptic ulcers, with 1% proceeding to gastro-intestinal cancers. As hygiene and public health improve, the prevalence of infections decreases. In successive birth cohorts of the Bristol Helicobacter project, cancer incidence has decreased correspondingly with H. pylori infections (Harvey et al, 2002; Lane et al, 2002). This progression supports the general relationships of the early-life infectious load to later mortality in successive cohorts (Fig. 2.8) and is a benchmark example of by-stander effects in infections (Queries 1 and 2, Section 1.1). The oncogenicity of H. pylori arises from chronic localized inflammatory responses around the mucosa to which it attaches extracellularly (Penta et al, 2005). 154 The Biology of Human Longevity
  • Mucosal cell proliferation is stimulated. Infiltrating macrophages and neutrophils produce promutagenic ROS that damage epithelial cells, in association with increased oxidative DNA damage, as assayed by intestinal 8-OHdG (Farinati et al, 2003). As further proof of the inflammation-oxidative damage link, treatments that eradicated H. pylori also decreased tissue level of 8-OHdG (Farinati et al, 2004). As expected from the increased intestinal proliferation, intestinal telomere DNA is also shortened in H. pylori infections, even without local metaplasia (Kuniyasu et al, 2003). There are instructive genetic interactions of host cytokine alleles with virulence factors of H. pylori. For example, cancer risk was 10-fold higher by the combined presence of a high-risk H. pylori virulence factor and high-risk IL-1 allele than in the opposite low-risk combination (Figueiredo et al, 2002). Anti-inflammatory NSAIDs, which inhibit COX-2, are protective in H. pylori infections (more on this in the next section). H. pylori infections are strong examples of inflammation-induced oxidative damage (Query II). Inflammatory bowl disease is among the top three main risk conditions for colorectal cancer. After 7 y of exposure to inflammatory bowl disease, the incidence of colorectal cancer increases by 0.5–1%/y (Itzkowitz and Yio, 2004). The area of the colon surface involved in inflammation also increases the risk of colorectal cancer, giving an example of bystander damage with dose effects from time and target area (Section 1.4). Oxidant stress from inflammation is a leading mechanism in colorectal cancer. Chromosomal instability, mutational loss of function in the APC and p53 genes, and induction of COX-2 and NO synthase are typical. Consistent with the inflammation hypothesis, aspirin and NSAIDs reduce the risk of colorectal cancer (Section 2.10.2). Virus-induced inflammation is also linked to liver cancer, which ranks sixth in the world and third in the U.S. as causes of death (Parkin et al, 2005). Most liver cancers, 75% worldwide, are caused by hepatitis B and C viruses (HBV, HBC). Each infection increases the risk about 20-fold, while co-infection may increase the risk >100-fold (Donato et al, 1998). A substantial minority (5–20%) of HBV infections progresses from cirrhosis, within 5 y of diagnosis, to hepatocellular carcinoma (Imperial, 1999; Marcellin et al, 2005). HBV-induced cancer is associ- ated with increased inflammatory expression: Although the acute phase responses during HBV were not reported in detail, IL-6 elevation correlates with the clinical severity (Tangkijvanich et al, 2000). Tumorigenesis depends on Hepatitis-Bx (HBx), a virally encoded signaling peptide that stimulates hepatocyte proliferation by activating NF-κB after viral integration. Hbx also facilitates metastasis by induc- ing matrix remodeling through matrix metalloproteinases and COX-2 (Lara-Pezzi et al, 2002). These latter inflammatory responses are all-too-familiar in atheromas (Section 1.5.3.1). COX-2 inhibitors suppress cell invasion in experimental models (Lara-Pezzi et al, 2002), but their efficacy has not been reported for HBV or HBC. These examples of H. pylori and hepatitis virus in inflammation and cancer merely scratch the surface of a growing literature that links chronic inflammation to cancer and that shows promise for anti-inflammatory drugs (see below). Infections, Inflammogens, and Drugs 155
  • 156 The Biology of Human Longevity 2.9.2. Smoking and Lung Cancer Smoking is the most robust example of an external inflammogen (bioaerosol) that is not infectious and that causes acceleration of mortality and premature mor- tality from multiple causes. Lung cancer was extremely rare before the advent of popular smoking beginning in the early 20th century. In Richard Doll’s classic lon- gitudinal study of British physicians since 1951, smokers have a risk that increases in proportion to the numbers of ‘pack-years.’ In contrast, those avoiding contact with cigarette smoke have negligible risk (Fig. 2.11) (Doll et al, 2004; Doll et al, 2005; Peto R et al, 2000; Vineis et al, 2004). For lifetime use, the risk of death from lung cancer is 5% in men and 10% in women. Smokers who quit retain the same risk for lung cancer indefinitely; e.g., men quitting at 60 had stabilized 10% during the remainder of their lives. These powerful dose-response relationships in smoking and cancer define a paradigm for environmental exposures that may extend to environmental factors in cancers in general. Besides the lung, smoking also increases the risk of cancer elsewhere in the larynx, stomach, liver, pancreas, and bladder. However, prostate cancer is only weakly, if at all, related to smoking (Vineis et al, 2004). Cancer 15 10 5 0 45 A 55 65 75 Y Cumulativerisk% Continued smoking Stopped age 50 Stopped age 30 Never smoked FIGURE 2.11 For legend see page 157.
  • risk in smokers may be subject to genetic variations in detoxifying enzymes, DNA repair (Wu X. et al, 2004), and apoE4 (Fig. 5.18). Smoking also increases the risk of vascular disease events, but the dose-response to pack-years is less clear. It is not known how tobacco smoke promotes, directly or indirectly, carcinogenesis in enteric organs that are not in direct contact with the airways. Infections, Inflammogens, and Drugs 157 40 B 9080706050 Y 0 20 40 60 80 100 0 20 40 60 80 100 %survivalafter60%survivalafter60 Born 1900-1930 Doctors and smoking Smokers Non-smokers 82 45 13 1 68 31 5 Smokers Non-smokers 71 32 5 85 65 26 2 Born 1851-1899 FIGURE 2.11 Smoking. A. Cumulative risk of death from lung cancer in the UK by age of smoking cessation since 1950. From national data combined with two case control studies. (Redrawn from Peto R et al, 2000.) B. Survival and smoking in British doctors. The smaller impact in doctors born before 1900 may reflect few pack-years. Note the greater life expectancy and percent surviving to age 90 in non-smokers born after 1900. (Redrawn from Doll et al, 2005.)
  • 2.10. PHARMACOPLEIOTROPIES IN VASCULAR DISEASE, DEMENTIA, AND CANCER I suggest the term ‘pharmacopleiotropy’ to represent multiple domains of drug effects with overlapping specificities. Many drugs influence biochemical and cell systems far beyond the original targets. Negative or adverse effects of drugs are well recognized, as in gastric bleeding from aspirin. However, unexpected pos- itive pharmacopleiotropies are emerging for drugs that protect against heart dis- ease which may also reduce the risk of cancer and of AD (Table 2.2). These cross-over effects are consistent with shared anti-inflammatory effects of diverse drugs in heart disease and with evidence for the pervasiveness of inflammatory processes in “normal” aging and in Alzheimer and vascular disease. Moreover, pharmacopleiotropies are consistent with genetic pleiotropies of apoE4 as a shared risk factor in Alzheimer and vascular disease and with the shared risk factors from the environment and lifestyle (Section 5.7). 2.10.1. Anti-inflammatory and Anti-coagulant Drugs The remarkable possibility can be considered that a cluster of major degenerative diseases is attenuated by the widely used drugs that were developed for heart dis- ease. Meta-analyses agree in the shared efficacy of quite different drugs in reduc- ing the risk of vascular events, certain cancers, and possibly Alzheimer disease, with effects in the broad range of 10–60% (Table 2.2). No single study can suffice, and there are many divergences between indications of efficacy from animal models and clinical effects. It is hard to interpret the drug literature on vascular dis- ease and dementia (Baron, 2003): Clinical end-points differ between studies, as do drug doses and durations. There is great heterogeneity in age, health status, eth- nicity, socioeconomics, and education. Those taking aspirin and other drugs often modify their lifestyles, which reduces their risks independently of the drug candi- date. Thus, the ‘possible benefits’ of the drugs in Table 2.2 should be considered a moving target of continuing critical evaluation for complex drug interactions to identify subgroups by risk and benefit for remaining life expectancy. The pharmacology of anti-inflammatory drugs distinguishes two broad groups: steroidal anti-inflammatory drugs (SAIDs) and non-steroidal anti-inflam- matory drugs (NSAIDs) (Hardman and Limbird, 2001; Vane and Botting, 2003). NSAIDs alter production of prostaglandins and thromboxanes, ‘isoprenoids’ that have diverse and broad roles in normal physiology and in disease, e.g. the thromboxane TXA2 enhances local platelet aggregation, while blockers of TXA2 can be protective against thrombosis. The ubiquitous isoprenoids are also referred to as ‘autocoids’ because they are produced from local membrane lipids. Arachidonic acid is an autocoid substrate of the cyclooxygenase enzymes, COX- 1, -2, and -3. COX-1 products mediate normal functions of gastric mucosa and kidney, as well as coagulation: COX-2 is induced during inflammatory reactions, 158 The Biology of Human Longevity
  • Infections, Inflammogens, and Drugs 159 e.g., by LPS and other endotoxins. COX-1 and COX-2 are coded by distinct genes, whereas COX-3 is a new splicing variant of COX-1 with overlapping func- tions (Botting, 2003; Vane et al, 2003). PI3-K, of the ‘insulin/IGF-1 metabolic longevity’ pathways (Fig. 1.3), mediates many NSAIDs’ actions. For example, PI3-K is required for induction of COX-2 by endotoxin (LPS) in mesangial cells (Sheu et al, 2005). Steroidal anti-inflammatory drugs, such as dexamethasone and prednisolone (synthetic glucocorticoids), act via specific receptors to modulate subcellular pathways that are in part distinct from those regulated directly by NSAIDs. Glucocorticoids have metabolic effects during chronic treatments that differ from NSAIDs. However, there are notable convergences; e.g., the COX-2 gene pro- moter has glucocorticoid response elements (GRE), which enable repression by cortisol and synthetic glucocorticoids (Kramer, 2004; Santini et al, 2001). TABLE 2.2 Possible Benefits of Adult-Onset Degenerative Diseases by Aspirin, NSAIDs, and Statins Aspirin NSAIDs Statins cancer colorectal & esophageal +2 +2 +/− bladder, breast, ovary, prostate +/− +/− +/− non-Hodgkin lymphoma −1 −1 neurodegenerative diseases Alzheimer disease + + +/− Parkinson + + vascular disease events myocardial infarct +2 −2 +2 stroke +/− −1 +2 +2, consistent benefit across many studies; +1, possible benefits according to initial observations; +/−, possible benefits, but not consistent; −1, possible adverse effects; −2, definite adverse effects. Other references in Section 2.1. cancer: aspirin and NSAIDs (Baron, 2003; Bosetti et al, 2002; Herendeen and Lindley, 2003; Perron et al, 2003; Meric et al, 2006); non-Hodgkin lymphoma (Cerhan, 2003). statin benefits to cancer are in Phase II trials with various tumors and are being considered for adjunct chemotherapy and chemopreventives (Chan et al, 2003; Etminan et al, 2003). neurodegenerative diseases: Alzheimer (Breitner, 2003; Etminan et al, 2003) Parkinson (Chen et al, 2003) vascular disease events: myocardial infarct: aspirin (Antithrombotic Trialists’ Collaboration, 2002; Eidelman et al, 2003; Bartolucci and Haward, 2006) statins (Fonarow and Watson, 2003; Heart Protection Collaborative Group, 2002; Law et al, 2003; Mostaghel and Waters, 2003; Wald and Law, 2003) stroke: aspirin (Bartolucci and Howard, 2006) NSAIDs (Kearney et al, 2006) statins (Amarenco et al, 2006; Endres and Laufs, 2006).
  • Of the NSAIDs, aspirin (acetylsalicylic acid) has been long known as an anal- gesic and antipyretic at low doses and is the mostly widely taken drug world- wide (Vane et al, 2003). Aspirin has anti-coagulant activities that reduce the risk of vascular events. At 160 mg/d, aspirin doubles the clotting time by irreversibly inhibiting platelet COX-1, which makes TXA2 from arachidonic acid, an omega-6 polyunsaturated fatty acid (PUFA). Aspirin effects on clotting last up to 10 days, the life span (t1⁄2 ) of blood platelets. Higher doses of aspirin are used for rheu- matic inflammatory diseases, but often cause gastric bleeding due to blockade of COX-1 in the gastric mucosa. However, even after its acetylation by aspirin, COX-2 activity persists for certain PUFA ω-3 and ω-6 substrates, some of which are anti-inflammatory, e.g., 15 epi-lipoxin A4 (Arita et al, 2005). Ibuprofen and naproxen are non-selective for COX-1 and-2 (Cryer and Feldman, 1998). Other NSAIDs were developed to target COX-2 without inhibiting COX-1, effectively reducing inflammatory responses with fewer gastric side effects, e.g., celecoxib, naproxen, and rofecoxib. COX-2 and prostaglandin production are also inhibited by synthetic glucocorticoids, as noted above. The NSAIDs field is in turmoil because of conclusive evidence for increased risk of vascular events (Hippisley-Cox and Coupland, 2005; Juni et al, 2004; Levesque et al, 2005). For example, a population-based case-control study in Finland showed that NSAIDs users had 40% higher risk of a myocardial infarct (OR, 1.40, 95% CI, 1.33–1.48) (Helin-Salmivaara et al, 2006). The various types of COX-2 inhibitors had similar effects. An underlying mechanism may be that inhibiting COX-2 may induce both proinflammatory and prothrombotic homeo- static compensatory responses (Doux et al, 2005). Homeostatic responses would be expected because of the importance of COX-2 to many normal cellular func- tions, e.g., celecoxib induced COX-2 in rat spinal cord (Hsueh et al, 2004). Moreover, due to their short half-lives in circulation (e.g., t1⁄2 11 h, celecoxob), there could be prothrombotic transients in prostaglandin conversion in the declining phase before another dose is taken. Withdrawal from chronic aspirin in stable cardiac patients, for example, may increase the incidence of thromboses (Senior, 2003). These early observations merit full study. Statin drugs were designed to lower blood cholesterol by blocking a rate- limiting enzyme of cholesterol synthesis (HGM-CoA reductase). In addition, statins have many effects on inflammation (Balk et al, 2003; Halcox and Deanfield, 2004l; Kwak et al, 2003; Schonbeck and Libby, 2004; Stuve et al, 2003), which may account for vascular benefits not directly linked to blood lipids. Cholesterol- independent pleiotropic effects of statins include the lowering of blood CRP (Albert et al, 2001), inflammatory cell infiltration and cell death (Wierzbicki et al, 2003), and impairing of lymphocyte proliferation (Palinski and Tsimikas, 2002b). Some statin effects result directly from the decrease of mevalonate from the inhi- bition of HMG CoA reductase. Intracellular mevalonate is a precursor to the iso- prenoids (farnesyl pyrophosphate, geranylgeranyl) that modulate enzymes with links to inflammation and vascular smooth muscle cell proliferation, e.g., the Ras and Rho kinases (Endres and Laufs, 2006; Kamiyama et al, 2003). PI3K/Akt is also 160 The Biology of Human Longevity
  • involved in the induction of nitric oxide synthase by statins (Walter et al, 2004). Statins also increase circulating endothelial progenitor cells (EPCs) (Urbich and Dimmeler, 2005; Vasa et al, 2001a,b), which are implicated in vascular repair (Sections 1.5.3.4 and 2.5.2) and may modulate EPC cell senescence by suppress- ing Chk2, a DNA damage checkpoint kinase induced by telomere dysfunction (Spyridopoulos et al, 2004). Cardioprotective effects of NSAIDs and statins involve neovascularization (Vagnucci and Li, 2003), which may be important to both AD neuritic plaques and in vascular atheromas (Chapter 1, Table 1.3). 2.10.2. Aspirin and Other NSAIDs Aspirin and NSAIDs have well established benefits to risks of heart attack and prob- ably to stroke. In the huge Anti-thrombotic Trialists’ Collaboration, aspirin and anti- platelet drugs collectively reduced vascular events: 25% risk reduction for myocardial infarction (primary or recurrent) and 11% reduction of stroke; aspirin at a low dose (75–150 mg/day) reduced risk by 32% (Antithrombotic Trialists’ Collaboration, 2002). Low dose aspirin in other studies reduced stroke by 13–16%. This net figure represents a greater reduction in thrombotic strokes than the increase of hemor- rhagic stroke in aspirin users (van Gijn and Algra, 2002; Wald and Law, 2003). Some cancer risks may benefit from aspirin and NSAIDs (Baron, 2003; Meric e